Araceli Diez-Fraile

Life and death of female gametes during oogenesis and folliculogenesis

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.

Age-associated differential microRNA levels in human follicular fluid reveal pathways potentially determining fertility and success of in vitro fertilization.

Abstract Reproductive life span and fertility have been shown to depend on successful early folliculogenesis, which involves cell-to-cell communication and the concerted regulation of gene expression at both the oocyte and granulosa cell levels. Recently, micro RNAs (miRNAs) were identified as fine-tuners of gene expression. Here, we report that miRNAs can readily be detected within membrane-enclosed vesicles of human follicular fluid. MiRNA expression profiling of the follicular fluid of younger (< 31 years) and older (> 38 years) women revealed a set of four differentially expressed miRNAs. The predicted targets of these miRNAs are clearly enriched in genes involved in heparan-sulfate biosynthesis, extracellular matrix–receptor interaction, carbohydrate digestion and absorption, p53 signaling, and cytokine–cytokine-receptor interaction. Several of these pathways have been reported to be determinants of fertility, suggesting that this set of miRNAs and their respective targets should be evaluated in relation to reproductive aging and assisted reproduction.

Regulation of adhesion molecules on circulating neutrophils during coliform mastitis and their possible immunomodulation with drugs

Fast neutrophil diapedesis has been demonstrated to be critical in coliform mastitis and is determining for the severity of infection. Leukocyte adhesion molecules play a pivotal role in neutrophil recruitment. Two families of cell surface proteins help to regulate the adherence of neutrophils to vascular endothelium: selectins and beta(2)-integrins. Both classes of leukocyte adhesion molecules are reviewed in the context of their dynamic expression around parturition and during acute coliform mastitis. Their potential modulation by commonly used drugs and the therapeutic implications during acute coliform mastitis are also discussed.

Persistence of Yersinia ruckeri in trout macrophages.

Yersinia ruckeri is the etiological agent of enteric redmouth disease, a systemic infection which mainly affects salmonids. Although this important freshwater pathogen was discovered in 1966, little is known about its virulence mechanisms. In the present study, the interactions with rainbow trout head kidney macrophages were investigated. In vitro experiments were performed to measure uptake, intracellular survival, respiratory burst response and macrophage viability after exposure to Y. ruckeri. Additionally, the fate of Y. ruckeri in the head kidney after immersion infection was studied in vivo. Results show that Y. ruckeri induced the production of reactive oxygen species and that this response peaked at around 3 h after exposure. Despite these toxic substances, Y. ruckeri is able to survive in vitro inside trout macrophages for at least 24 h. Transmission electron microscopy demonstrated that Y. ruckeri bacteria are sequestered in autophagocytic compartments without fusion with primary lysosomes. Inside these compartments, bacteria were capable of replicating. Immersion infection of juvenile rainbow trout resulted in steadily increasing numbers of bacteria in the head kidney over time. As the infection progressed, Y. ruckeri shifted from a predominantly extracellular phase during the first week after infection to an intracellular phase inside the host macrophages from day 7 onwards. In conclusion, this study clearly demonstrates the capacity of Y. ruckeri to survive in rainbow trout macrophages in vitro as well as in vivo, confirming its facultative intracellular nature.

Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils.

The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist). Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression. A combination of IPN and DX elicited a synergistical decrease of the CD11b expression. Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not. The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation. Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation. This inhibition is probably related to a decrease in TNF-alpha production. A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.

Sympathoadrenal and immune system activation during the periparturient period and their association with bovine coliform mastitis. A review

Summary Increased incidence of clinical mastitis in high‐yielding cows during early lactation has been attributed to a depressed functional capacity of the immune system. Sympathoadrenal factors are known to play an important role in modulating the host susceptibility and resistance to infectious diseases. Of primary importance in combating acute intramammary infections are polymorphonuclear leukocytes (PMN), as they represent one of the early lines of immunological defense. The release of stress hormones at parturition and during the first weeks of lactation has been proposed to partly contribute to the impaired function of PMN. Here, we summarize the current understanding of the stress‐induced peripheral effectors, i.e. the limbs of the sympathetic system and the hypothalamic‐pituitary‐adrenal axis, on PMN function around parturition and during coliform mastitis. The questions as to whether and how stress induced secretion of glucocorticoids and catecholamines might affect the lactating dairy cows udder health will be addressed.

The Calcium-Sensing Receptor as a Regulator of Cellular Fate in Normal and Pathological Conditions

The calcium-sensing receptor (CaSR) belongs to the evolutionarily conserved family of plasma membrane G protein-coupled receptors (GPCRs). Early studies identified an essential role for the CaSR in systemic calcium homeostasis through its ability to sense small changes in circulating calcium concentration and to couple this information to intracellular signaling pathways that influence parathyroid hormone secretion. However, the presence of CaSR protein in tissues is not directly involved in regulating mineral ion homeostasis points to a role for the CaSR in other cellular functions including the control of cellular proliferation, differentiation and apoptosis. This position at the crossroads of cellular fate designates the CaSR as an interesting study subject is likely to be involved in a variety of previously unconsidered human pathologies, including cancer, atherosclerosis and Alzheimers disease. Here, we will review the recent discoveries regarding the relevance of CaSR signaling in development and disease. Furthermore, we will discuss the rational for developing and using CaSR-based therapeutics.

Expression of calcium-sensing receptor in quail granulosa explants: a key to survival during folliculogenesis.

This study investigated the potential role of the calcium‐sensing receptor (CaR) in mediating survival of granulosa cells (GCs) in follicles of the Japanese quail (Coturnix coturnix japonica). Immunoreactivity of CaR was shown in GCs of quail preovulatory follicles as well as in the remnants of the GC layer after ovulation. Conversely, the CaR could not be detected by immunocytochemistry in the granulosa of smaller undifferentiated follicles. The presence of CaR in follicles destined to ovulate was confirmed by immunoblot and the receptor was identified as a protein of 115–125 kDa. Addition of different CaR agonists to granulosa explants in culture for 24 hr caused inhibition of apoptosis elicited by gonadotropin withdrawal on its own or in combination with C8‐ceramide addition. Furthermore, R‐568, a specific, positive allosteric modulator of CaR, not only inhibited apoptosis but also increased GC number per viewing field in cultured granulosa explants. This observation could be attributed not to a rise in GC proliferation but to a more compact tissue structure, including a distinct distribution pattern of connexin‐43 gap junction proteins. Incubation in the presence of LY294002, a specific phosphatidylinositol‐3 kinase inhibitor, increased GC apoptosis, indicating that this pathway is involved in GC survival signaling. However, LY294002‐induced apoptosis was considerably attenuated by incubation with R‐568, indicating that other pathways might be major contributors to the survival mediated by CaR agonists. We provide direct evidence for the presence of CaR in preovulatory granulosa explants and suggest a pivotal role for CaR in follicle selection. Anat Rec, 2010.

Long non-coding RNAs in pluripotent stem cell biology

Pluripotent stem cells are defined by their unlimited self-renewal capacities and potential to differentiate into any cell lineage. Many crucial determinants for the induction and maintenance of this pluripotent state have been identified. Long non-coding RNAs have recently emerged as key regulators of pluripotent stem cells and have enhanced our understanding of their potential functions in tissue regeneration. This review provides an overview of recent important insights into the roles of long non-coding RNAs as regulators and markers of pluripotency.

Optimizing Multiple Immunostaining of Neural Tissue

Interference from autofluorescence is one of the major problems in immunofluorescence analysis of neural tissue. This difficulty is experienced even with the use of laser-scanning microscopy, which can eliminate out of focus light but not the undesirable tissue-specific autofluorescence that is in focus. Therefore, we will center our discussion on different strategies to minimize neural tissue autofluorescence. The two greatest sources of autofluorescence discussed here are those induced by aldehyde fixation and by the fluorescent pigment lipofuscin, which accumulates with age in the cytoplasm of cells of the central nervous system. Finally, it is important to determine whether any treatments used to minimize autofluorescence might quench specific fluorescent compounds used to label the proteins of interest. We present the results of multi-label fluorescence immunostaining of cryostat sections of 20 μm of the human inferior colliculus. We conclude that the use of chemical agents to reduce autofluorescence and quench lipofuscin-specific fluorescence helps to maximize the fluorescent signal-to-noise ratio in immunocytochemical studies of fixed neural tissues.