Effect of expression of estrus and treatment with GnRH on pregnancies per AI in beef cattle synchronized with an estradiol/progesterone-based protocol.

Three experiments evaluated the effects of expression of estrus and gonadotropin releasing hormone (GnRH) treatment on pregnancies per AI (P/AI) in beef cattle that were treated with an estradiol/progesterone (P4)-based protocol for fixed-time artificial insemination (FTAI). In Experiment 1, 20 non-lactating beef cows were treated with 2 mg estradiol benzoate (EB) and an intravaginal device containing 0.5 g of P4. Seven days later, P4 devices were removed and all cows received prostaglandin F2 alpha (PGF2α) and 0.5 mg estradiol cypionate (ECP). Estrus was detected using tail paint and cows that did not show estrus by 48 h after P4 device removal were randomized to receive GnRH or no treatment. Ovulation, as determined by ultrasonography, occurred earlier in cows that showed estrus (68.0 ± 2.5 h) than in cows that did not (82.0 ± 2.1 h, P < 0.05). Furthermore, cows that received GnRH ovulated earlier (78.0 ± 2.6 h) than those that did not (86.0 ± 2.0, P < 0.05). Experiment 2 determined whether expression of estrus and the administration of GnRH to animals that did not show estrus increased P/AI. Non-lactating beef cows and heifers (n = 1356) were treated as in Experiment 1 (P4 device removal, PGF2α and ECP administration on Day 7) or extended until Day 8. All animals in estrus by 48 h after P4 device removal were inseminated and those not showing estrus received GnRH or no treatment and were FTAI 8 h later (i.e., at 56 h). P/AI were greater (P < 0.01) in animals that were observed in estrus by 48 h (56.4%) than in those that did not show estrus (46.5%). Likewise, animals that did not show estrus but were treated with GnRH had greater P/AI (53.8%, P < 0.04) than those that did not receive GnRH (37.9%). Experiment 3 was designed to determine the effect of delaying GnRH treatment to the time of FTAI (at 56 h) in cows not showing estrus by 48 h after P4 device removal. Suckled beef cows (n = 969) were treated as in Experiment 1, except that all cows also received 400 IU of eCG at the time of P4 device removal on Day 7. Cows that showed estrus by 48 h or 56 h had greater P/AI (62.3%, P < 0.05) than those did not show estrus (51.5%). Furthermore, when cows that did not show estrus by 48 h were analyzed separately, P/AI were greater (P < 0.05) in those that received GnRH at 48 h and were FTAI by 56 h (64.9%) than in those that received GnRH concurrent with FTAI by 56 h after device removal (54.6%). In summary, expression of estrus was associated with earlier ovulations and resulted in greater P/AI in cows and heifers treated with an estradiol/P4-based protocol for FTAI. Furthermore, GnRH treatment in animals that did not show estrus hastened the time of ovulation and increased P/AI.

El uso de estrategias de afrontamiento del estrés en personas con discapacidad intelectual / Coping strategies among people with intellectual disability

Durante un tiempo se consideró que las personas con discapacidad intelectual (DI) no se veían afectadas negativamente por el estrés y por ello no se incluían en los estudios de afrontamiento del estrés. En este trabajo se aplica la adaptación española del Inventario de Estrategias de Afrontamiento (CSI) a una muestra de 262 adultos, entre 20 y 74 años, 108 con DI y 154 sin DI, de Burgos (España). Se observan diferencias significativas en las personas con DI en relación con la edad: en apoyo social, los menores de 40 años puntúan más alto que los mayores de 50. En las personas sin DI, las mujeres tienden a utilizar significativamente más las estrategias centradas en la búsqueda de apoyo (M = 15.05, DT = 6.21) y en la liberación de emociones (M = 12.16, DT = 4.3) que los hombres (M = 10.00, DT = 6.21; M = 8.29, DT = 5.26), pero estos recurren más a la estrategia de retirada social (M = 8.09, DT = 5.19) que las mujeres (M = 5.05, DT = 3.82). Las personas con DI utilizan menos las estrategias de resolución de problemas, reestructuración cognitiva y apoyo social que las personas sin DI

For long it was thought that people with intellectual disabilities (ID) were not adversely affected by stress and therefore were not included in stress research. In this paper, the Spanish adaptation of the Coping Strategies Inventory (CSI) was applied to 262 adults aged between 20 and 74 years old, 108 with ID and 154 without ID from Burgos, Spain. People with ID showed significant aged-related differences in social support in relation to age, people over 40 scoring higher than people over 50. In the group of people without ID, women used more strategies focused on search for support (M = 15.05, SD = 6.21) and on emotion-release (M = 12.16, SD = 4.3) than men (M = 10.00, SD = 6.21; M = 8.29, SD = 5.26), but men resorted more to the social withdrawal strategy (M = 8.09, DT = 5.19) than women (M = 5.05, DT = 3.82). People with ID were found to use problem solving, cognitive restructuring, and social support strategies less frequently than people without ID

Detection of cells programmed to die in mouse embryos.

Programmed Cell Death (PCD) is a broad term used to describe a series of events that culminate in the death of specific cells. In the embryo it occurs at predictable stages and tissues. During mouse development, PCD is a mechanism to preserve the homeostasis of the growing organism, and also is needed for the morphogenesis of a variety of structures. Apoptosis or PCD type I shows a sequence of morphological and biochemical changes such as plasma membrane blebbing, increase in mitochondrial membrane permeability, caspase activation, chromatin condensation, and phagocytosis. Many of these changes can be used to determine the occurrence of apoptosis in different type of samples. For example, apoptosis has been visualized in whole embryos and tissue sections using vital dyes, and by detection of degraded DNA or active caspases. In the present report, we compare these methods during the course of interdigital cell death in the mouse limbs. We discuss which method is the most suitable to detect a particular stage of apoptosis, which in some cases may be relevant for the interpretation of data. We detail combined protocols to observe mRNA expression or protein and cell death in the same tissue sample. Furthermore, we discuss some of the methodological problems to analyze autophagic cell death or PCD type II during embryo development.

Chemical activation of RARß induces post-embryonically bilateral limb duplication during Xenopus limb regeneration.

The anuran amphibian Xenopus laevis can regenerate its limbs for a limited time during the larval stage, while limbs are still developing. Using this regeneration model, we evaluated the proximo-distal blastema cell identity when endogenous retinoids were increased by CYP26 inhibition or when RAR-specific agonists altered RA signaling. Simultaneous proximo-distal and antero-posterior limb duplications were generated, and the RAR-specific agonist can modify blastema identity after amputation, because chemical activation of RARß produced bilateral hindlimb duplications that resulted in a drastic duplication phenotype of regenerating limbs.

Full regeneration of the tribasal Polypterus fin.

Full limb regeneration is a property that seems to be restricted to urodele amphibians. Here we found that Polypterus, the most basal living ray-finned fish, regenerates its pectoral lobed fins with a remarkable accuracy. Pectoral Polypterus fins are complex, formed by a well-organized endoskeleton to which the exoskeleton rays are connected. Regeneration initiates with the formation of a blastema similar to that observed in regenerating amphibian limbs. Retinoic acid induces dose-dependent phenotypes ranging from inhibition of regeneration to apparent anterior-posterior duplications. As in all developing tetrapod limbs and regenerating amphibian blastema, Sonic hedgehog is expressed in the posterior mesenchyme during fin regeneration. Hedgehog signaling plays a role in the regeneration and patterning processes: an increase or reduction of fin bony elements results when this signaling is activated or disrupted, respectively. The tail fin also regenerates but, in contrast with pectoral fins, regeneration can resume after release from the arrest caused by hedgehog inhibition. A comparative analysis of fin phenotypes obtained after retinoic acid treatment or altering the hedgehog signaling levels during regeneration allowed us to assign a limb tetrapod equivalent segment to Polypterus fin skeletal structures, thus providing clues to the origin of the autopod. We propose that appendage regeneration was a common property of vertebrates during the fin to limb transition.

Molecular control of cell differentiation and programmed cell death during digit development.

During the hand plate development, the processes of cell differentiation and control of cell death are relevant to ensure a correct shape of the limb. The progenitor cell pool that later will differentiate into cartilage to form the digits arises from undifferentiated mesenchymal cells beneath the apical ectodermal ridge (AER). Once these cells abandon the area of influence of signals from AER and ectoderm, some cells are committed to chondrocyte lineage forming the digital rays. However, if the cells are not committed to chondrocyte lineage, they will form the prospective interdigits that in species with free digits will subsequently die. In this work, we provide the overview of the molecular interactions between different signaling pathways responsible for the formation of digit and interdigit regions. In addition, we briefly describe some experiments concerning the most important signals responsible for promoting cell death. Finally, on the basis that the interdigital tissue has chondrogenic potential, we discuss the hypothesis that apoptotic-promoting signals might also act as antichondrogenic factors and chondrogenic factors might operate as anti-apoptotic factors.

Smad8 is expressed in the anterior necrotic zone: evidence for a role of bone morphogenetic proteins/SMAD signaling in the activation of a molecular cascade that culminates in cell death.

Bone morphogenetic proteins (BMPs) play a crucial role in programmed cell death (PCD), a biological process required for the sculpturing of the embryonic limbs. However, it is unknown if BMP signaling directly promotes cell death, or if it induces a molecular cascade that culminates in cell death. Given that Smad8, which encodes one component of BMP signaling, is expressed during the regression of interdigital tissue and responds to BMPs, we presumed that it may be expressed in other cell death areas during chick limb development such as the anterior and posterior necrotic zones (ANZ and PNZ). The present study found that the Smad8 expression pattern in the anterior mesoderm of the hindlimb is very similar to that observed in limbs stained to detect cell death. Also, BMPs and retinoic acid, which act as apoptosis-promoting factors, induced expression of Smad8 before the onset of cell death, while sonic hedgehog protein, acting as a survival factor, inhibited Smad8 expression in the ANZ. However, although there was correlation between Smad8 expression patterns and PCD in the ANZ, phosphorylated forms of SMAD1/5/8 and TUNEL staining did not co-localize in dying cells. Interestingly, a short pulse of BMP was sufficient to trigger cell death. On the other hand, most dying cells were located in the avascular region, while many cells expressing Smad8 were located in the vascular region of the ANZ. These results suggest that BMPs mediated by SMAD signaling activate a molecular cascade that culminates in PCD.

Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death.

Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital 'necrotic' tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene (Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.

Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis.

During mammalian development, a pair of shelves fuses to form the secondary palate, a process that requires the adhesion of the medial edge epithelial tissue (MEE) of each shelf and the degeneration of the resulting medial epithelial seam (MES). It has been reported that epithelial-mesenchymal transformation (EMT) occurs during shelf fusion and is considered a fundamental process for MES degeneration. We recently found that cell death is a necessary process for shelf fusion. These findings uncovered the relevance of cell death in MES degeneration; however, they do not discard the participation of other processes. In the present work, we focus on the evaluation of the processes that could contribute to palate shelf fusion. We tested EMT by traditional labeling of MEE cells with a dye, by infection of MEE with an adenovirus carrying the lacZ gene, and by fusing wild-type shelves with the ones from EGFP-expressing mouse embryos. Fate of MEE labeled cells was followed by culturing whole palates, or by a novel slice culture system that allows individual cells to be followed during the fusion process. Very few labeled cells were found in the mesenchyme compartment, and almost all were undergoing cell death. Inhibition of metalloproteinases prevented basal lamina degradation without affecting MES degeneration and MEE cell death. Remarkably, independently of shelf fusion, activation of cell death promoted the degradation of the basal lamina underlying the MEE ('cataptosis'). Finally, by specific labeling of periderm cells (i.e. the superficial cells that cover the basal epithelium), we observed that epithelial triangles at oral and nasal ends of the epithelial seam do not appear to result from MEE cell migration but rather from periderm cell migration. Inhibition of migration or removal of these periderm cells suggests that they have a transient function controlling MEE cell adhesion and survival, and ultimately die within the epithelial triangles. We conclude that MES degeneration occurs almost uniquely by cell death, and for the first time we show that this process can activate basal lamina degradation during a developmental process.

Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid.

The actual role of programmed cell death (PCD) in embryonic processes and the extrinsic signals that define the death fate in developing cells are still poorly understood. Here, we show that during secondary palate shelf fusion in the mouse, PCD appeared in the medial edge epithelia (MEE) of the anterior region only after shelf contact. Contact was necessary for efficient cell death activation in the MEE. However, exogenous all-trans-retinoic acid (RA) increased cell death independently of contact. Competence to induce cell death by contact or by RA exposure was obtained when the MEE were close to touch. Endogenous RA is a relevant regulator of the secondary palate PCD since this was reduced by a retinol dehydrogenase inhibitor and an RAR specific antagonist. Bmp-7 expression was positively regulated by RA. However, BMP-7 was unable to activate cell death within the palate tissue and NOGGIN, a natural BMP antagonist, did not block PCD. Reduction of PCD at the MEE directly with a caspase inhibitor or by inhibiting retinol dehydrogenase resulted in unfused palate shelves, but adhesion was not affected. In contrast, exogenous RA also blocked fusion, but in this situation the increased cell death within the MEE appeared to affect adhesion, thereby causing cleft palate in vivo.