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Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1-ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Besides its fibrinolytic function, the plasminogen–plasmin (PLG–PLA) system is also involved in fertilisation, where plasminogen activators bind to plasminogen to produce plasmin, which modulates sperm binding to the zona pellucida. However, controversy exists, depending on the species, concerning the role of the different components of the system. This study focused its attention on the role of the PLG–PLA system on fertilisation in the mouse with special attention to tissue plasminogen activator (tPA). The presence of exogenous plasminogen reduced in vitro fertilisation (IVF) rates and this decline was attenuated by the presence of plasmin inhibitors in combination with plasminogen. The incubation of spermatozoa with either oocytes or cumulus cells together with plasminogen did not change the acrosome reaction but reduced the number of spermatozoa attached. When spermatozoa from tPA−/− mice were used, the IVF rate decreased drastically, although the addition of exogenous tPA during gamete co-incubation under in vitro conditions increased fertilisation success. Moreover, fertility could not be restored after in vivo insemination of tPA−/− spermatozoa in the female ampulla, although tPA−/− males were able to fertilise in vivo. This study suggests a regulatory role of the PLG–PLA system during fertilisation in the mouse with possible implications in human reproduction clinics, such as failures in tPA production, which could be partially resolved by the addition of exogenous tPA during IVF treatment.

Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.

Background:Asthma is a multifactorial condition where patients with identical clinical diagnoses do not have the same clinical history or respond to treatment. This clinical heterogeneity is reflected in the definition of two main endotypes. We aimed to explore the metabolic and microbiota signatures that characterize the clinical allergic asthma phenotype in obese children.Methods:We used a multi-omics approach combining clinical data, plasma and fecal inflammatory biomarkers, metagenomics, and metabolomics data in a cohort of allergic asthmatic children.Results:We observed that the obese allergic asthmatic phenotype was markedly associated with higher levels of leptin and lower relative proportions of plasma acetate and a member from theClostridialesorder. Moreover, allergic children with a worse asthma outcome showed higher levels of large unstained cells, fecal D lactate and D/L lactate ratio, and with a higher relative proportion of plasma creatinine and an unclassified family member from the RF39 order belonging to the Mollicutes class. Otherwise, children with persistent asthma presented lower levels of plasma citrate and dimethylsulfone.Conclusion:Our integrative approach shows the molecular heterogeneity of the allergic asthma phenotype while highlighting the use of omics technologies to examine the clinical phenotype at a more holistic level.

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) proteincoated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 mg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase. © 2010 Society for Reproduction and Fertility.

A well-developed Holocene tufa system occurs in the headwaters of Las Parras River, Iberian Range (Teruel province). The longitudinal profile of the river shows two knickpoints downstream of two gorges known as Hocino de las Palomas and Hocino del Pajazo. The largest tufa build-ups in this area (49 m and 26 m thick) were found just downstream these two gorges and associated waterfalls. Barrage and cascade structures and facies suggest a rapid down-stream and vertical development of the tufa system. Between the knickpoints and upstream, the river valley displays a low-gradient (2%) and tufa deposits occur as laterally extensive terraces, with variable thickness (up to 8.2 m in El Batán) and fluvio-lacustrine tufa sequences associated to dammed areas. The Las Parras Tufa system represents a depositional environment including stepped fluvial systems with barrage-cascade and associated dammed areas separated by low gradient fluvial stretches. According to the chronological data, the development of Las Parras Holocene tufa occurred between 9.9 ky and 3.1 ky, with the highest growth rate from 5.5 to 3.1 ky, and a significant reduction of tufa activity during the last 2000 years. This evolution reflects the water availability in the area, and is coherent with changes in summer insolation during the Holocene., En el valle del río Las Parras (Cuenca alta del río Martín, Cordillera Ibérica turolense) aparecen importantes registros tobáceos holocenos. El perfil longitudinal del río muestra dos fuertes rupturas de pendiente coincidiendo con la salida de sendos estrechamientos conocidos como los hocinos de las Palomas y del Pajazo. En estos puntos se encuentran las acumulaciones tobáceas más potentes, 49 m en el Hocino de las Palomas y 26 m en el Hocino del Pajazo, con facies que indican un crecimiento rápido en un sistema de cascadas y barreras que se desarrollan vertical y lateralmente aguas abajo. El resto del tramo estudiado presenta pendientes suaves (2%) con depósitos tobáceos fluvio-lacustres, de morfología aterrazada de gran desarrollo longitudinal y menor potencia (hasta 8 m en El Batán), asociados con represamientos. El conjunto morfosedimentario corresponde a un sistema fluvial escalonado compuesto por cascadas y áreas represadas. Los datos cronológicos indican que las tobas holocenas del río Las Parras se formaron entre 9.9 y 3.1 ka, con un periodo de mayor desarrollo entre 5.5 y 3.1 ka. En los últimos 2000 años se produce un descenso brusco en la actividad tobácea en la zona que coincide con un periodo de mínima insolación estival y probablemente con una menor disponibilidad hídrica en la zona.

The unique geological framework of the "los Llanos de La Larri" (Ordesa-Monte Perdido National Park) facilitated the genesis of a relevant juxtaglacial sedimentary record during the last glacial phase in the central Pyrenees. In this study we conducted a geomorphological and outcrop survey, an electric tomography study and the definition of the main sedimentological units by means of the study of 3 cores up to 17 m long. The combined study of surface and underground data has allowed us to define a paleolacustrine sedimentary sequence more than 40 m thick and the geometry and nature of the units of the infilling sequence. Radiocarbon dating indicate that the minimum age of this deposit is 35 ky (BP) at 17 m depth, and the infilling of the basin ends at 11 ky BP., El singular contexto geológico de los llanos de La Larri (Parque Nacional de Ordesa-Monte Perdido, Pirineo Oscense) facilitó la formación de un importante complejo sedimentario de carácter yuxtaglaciar durante el último ciclo glaciar en el Pirineo central. En este estudio se ha realizado un análisis conjunto de cartografía geomorfológica, descripción de afloramientos, levantamiento e interpretación de perfiles de tomografía eléctrica y estudio de las principales unidades sedimentarias encontradas en 3 sondeos de hasta 17 m de profundidad. Los resultados han puesto de manifiesto la existencia de un importante paleolago con una potencia superior a 40 m, así como ha permitido definir la geometría del depósito y sus principales unidades. Las dataciones mediante radiocarbono de algunas muestras de superficie y subsuelo permiten afirmar que la sedimentación en el paleolago se inició antes de los 35 ka y finalizó, por la colmatación completa de su cuenca, hace unos 11 ka.

De las ocho campañas de excavación que se desarrollaron en el yacimiento arqueológico de Son Fornés, entre los años 1975 y 1988, se han estudiado 330 bordes de ánfora procedentes del área del Mediterráneo occidental, desde Sicilia, la península itálica, Ibiza, el Norte de África hasta las diferentes provincias romanas de Hispania. A partir de la adscripción tipológica y cronológica de cada pieza anfórica y de su cuantificación en el conjunto global de las ánforas documentadas en el yacimiento, se elabora un estudio sobre las relaciones y corrientes comerciales que se establecen entre el s.IV aC. y el s.I dC. y su coordinación con los hechos históricos más destacados de cada momento, integrando el registro arqueológico de un poblado de cultura talayótica de Mallorca en los acontecimientos históricos del Mediterráneo Occidental.