Cultiu de les cèl·lules epididimàries de "Sus domesticus": anàlisi estructural, funcional i proteòmic

En aquest treball s'han desenvolupat dos mètodes simples i ràpids pel cultiu de les cèl·lules epitelials de les tres regions de l'epidídim de Sus domesticus. Un es basa en el cultiu de fragments del túbul epididimari intactes durant 8 dies. L'altre mètode es basa en el cultiu de fragments del túbul epididimari digerits amb col·lagenasa que, després de 7 dies, donen lloc a la formació d'una monocapa de cèl·lules epitelials epididimàries que adquireixen el 90-100% de confluència després de 12-16 dies en cultiu. Aquestes cèl·lules es mantenen viables durant més de 60 dies en cultiu i no s'observa proliferació de cèl·lules no epitelials. Per determinar el nivell de conservació de les característiques epididimàries en els cultius s'ha analitzat l'estructura cel·lular, l'activitat de síntesi i secreció proteica, i el manteniment i maduració dels espermatozoides en cocultiu.

Estudi citològic i bioquímic del fluid epididimari de "Sus domesticus"

En aquest estudi s'ha determinat que al augmentar el ritme d'extraccions de semen es produeixen canvis en el patró d'absorció i secreció del fluid epididimari, que provoquen alteracions en la maduració epididimaria dels espermatozoides i un desenvolupament anòmal de la motilitat espermàtica. La concentració de glutamat i carnitina al fluid epididimari augmenten al llarg del conducte epididimari, alhora que la concentració de myo-inositol disminueix. El contingut de myo-inositol a l'interior dels espermatozoides disminueix, mentre que el contingut de glutamat augmenta a partir del caput distal i el contingut de carnitina no varia al llarg del conducte. S'ha determinat la presència de la ruta del poliol a l'epidídim de porcí. Els resultats obtinguts indiquen que la glucosa difon de la sang cap al fluid epididimari, és convertida a sorbitol per l'aldosa reductasa, i aquest sorbitol s'acumula al fluid luminal i és convertit a fructosa per l'acció de la sorbitol deshidrogenasa.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.

MS-based clinical proteomics: biomarker discovery in men’s cancer

Importance of biomarker discovery in men’s cancer diagnosis and prognosis Each year around 10,000 men in the UK die as a result of prostate cancer (PCa) making it the 3rd most common cancer behind lung and breast cancer; worldwide more than 670,000 men are diagnosed every year with the disease [1]. Current methods of diagnosis of PCa mainly rely on the detection of elevated prostate-specific antigen (PSA) levels in serum and/or physical examination by a doctor for the detection of an abnormal prostate. PSA is a glycoprotein produced almost exclusively by the epithelial cells of the prostate gland [2]. Its role is not fully understood, although it is known that it forms part of the ejaculate and its function is to solubilise the sperm to give them the mobility to swim. Raised PSA levels in serum are thought to be due to both an increased production of PSA from the proliferated prostate cells, and a diminished architecture of affected cells, allowing an easier distribution of PSA into the wider circulatory system.

Female chromosomes in cockerel ejaculates

We describe a polymerase chain reaction which amplifies part of the Eco RI repeat unit of the fowl W chromosome. The resulting 447 bp fragment enables DNA from female birds to be identified. The composition of this DNA is confirmed by a nested polymerase chain reaction which specifically amplifies a known internal 263 bp region in this fragment. Using this technique it is possible to follow the fate of female cells in male germline chimaeras. The polymerase chain reaction fragment can be traced in cells of the embryonic and hatchling gonad and in adult sperm implying that cells containing the W chromosome are capable of being processed through the avian testis.

Effect of media polarity on the photoisomerisation of substituted stilbene, azobenzene and imine chromophores

The influence of substituents and media polarity on the photoinducedE→Z geometrical isomerisation of the stilbene, azobenzene and N-benzylideneaniline chromophores has been compared and assessed. The efficiency of the process in all three systems is markedly dependent on the presence and characteristics of electron-donor and electron-acceptor substituents at the 4- and 4′-positions. The results are discussed in terms of relaxation of the E-excited singlet state. In the absence of a nitro substituent, relaxation to the S1 orthogonal state competes effectively with non-productive intramolecular electron transfer; in the presence of a nitro substituent, the T1 orthogonal state is formed from inter-system crossing. For systems with a 4-nitro and a 4′-electron-donor substituent, access to the triplet state is inhibited by polar solvents promoting formation of the inactive charge-transfer state from the S1 state, and no isomerisation is observed. Similar effects are observed in both solution and polymer films. Such variations in behaviour have important implications for the utilisation of the chromophores in nonlinear optical phenomena including photorefractivity.

A new parenthood paradigm for twenty-first century family law in England and Wales?

There are few other areas in family law where incongruence between the legal and social positions is as evident as that concerning parenthood. Recent cases involving lesbian couples and known sperm donors serve to highlight the increasing tension between the respective roles of biology, intention and functional parenting in the attribution of legal parental status. As both legislative and case-law developments have shown, intention is central in some circumstances, but not in others. The main claim of this paper is that this ad hoc approach leads to incoherent and unsatisfactory law: instead of striving to identify a status, what we are really looking to do is to identify the people who assume responsibility for a child. Drawing upon recent case-law, this paper explores how a conceptual reform of the law could result in a principled framework which would place formally recognised intention at the heart of parental status in order to reconnect legal duty with social reality for as many children and parents as possible. Moreover, it would ensure that parental status would not be dictated by the mode of conception of the child (natural or assisted). The analysis identifies the objectives of reform before proposing a new model which, while recognising the social importance of the biological parentage link, would reserve legal status for functional parenthood.

Do variations in substitution rates and male mutation bias correlate with life-history traits? A study of 32 mammalian genomes

Life-history traits vary substantially across species, and have been demonstrated to affect substitution rates. We compute genomewide, branch-specific estimates of male mutation bias (the ratio of male-to-female mutation rates) across 32 mammalian genomes and study how these vary with life-history traits (generation time, metabolic rate, and sperm competition). We also investigate the influence of life-history traits on substitution rates at unconstrained sites across a wide phylogenetic range. We observe that increased generation time is the strongest predictor of variation in both substitution rates (for which it is a negative predictor) and male mutation bias (for which it is a positive predictor). Although less significant, we also observe that estimates of metabolic rate, reflecting replication-independent DNA damage and repair mechanisms, correlate negatively with autosomal substitution rates, and positively with male mutation bias. Finally, in contrast to expectations, we find no significant correlation between sperm competition and either autosomal substitution rates or male mutation bias. Our results support the important but frequently opposite effects of some, but not all, life history traits on substitution rates. KEY WORDS: Generation time, genome evolution, metabolic rate, sperm competition.

Production of Bovine Hand-Made Cloned Embryos by Zygote-Oocyte Cytoplasmic Hemi-complementation

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; handmade cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Testicular traits as selection criteria for young Nellore bulls

The objective of this study was to evaluate the possible use of biometric testicular traits as selection criteria for young Nellore bulls using Bayesian inference to estimate heritability coefficients and genetic correlations. Multitrait analysis was performed including 17,211 records of scrotal circumference obtained during andrological assessment (SCAND) and 15,313 records of testicular volume and shape. In addition, 50,809 records of scrotal circumference at 18 mo (SC18), used as an anchor trait, were analyzed. The (co) variance components and breeding values were estimated by Gibbs sampling using the Gibbs2F90 program under an animal model that included contemporary groups as fixed effects, age of the animal as a linear covariate, and direct additive genetic effects as random effects. Heritabilities of 0.42, 0.43, 0.31, 0.20, 0.04, 0.16, 0.15, and 0.10 were obtained for SC18, SCAND, testicular volume, testicular shape, minor defects, major defects, total defects, and satisfactory andrological evaluation, respectively. The genetic correlations between SC18 and the other traits were 0.84 (SCAND), 0.75 (testicular shape), 0.44 (testicular volume), -0.23 (minor defects), -0.16 (major defects), -0.24 (total defects), and 0.56 (satisfactory andrological evaluation). Genetic correlations of 0.94 and 0.52 were obtained between SCAND and testicular volume and shape, respectively, and of 0.52 between testicular volume and testicular shape. In addition to favorable genetic parameter estimates, SC18 was found to be the most advantageous testicular trait due to its easy measurement before andrological assessment of the animals, even though the utilization of biometric testicular traits as selection criteria was also found to be possible. In conclusion, SC18 and biometric testicular traits can be adopted as a selection criterion to improve the fertility of young Nellore bulls.

Alternative mating tactics in dimorphic males of the harvestman Longiperna concolor (Arachnida: Opiliones)

Intense male-male competition for females may drive the evolution of male morphological dimorphism, which is frequently associated with alternative mating tactics. Using modern techniques for the detection of discontinuous allometries, we describe male dimorphism in the Neotropical harvestman Longiperna concolor, the males of which use their elongated, sexually dimorphic legs IV in fights for the possession of territories where females lay eggs. We also tested three predictions related to the existence of alternative mating tactics: (1) if individuals with relatively longer legs IV (majors) are more likely to monopolize access to reproductive resources, they are expected to remain close to stable groups of females more than individuals with relatively shorter legs IV (minors) do; (2) if minors achieve fertilization by moving between territories, they are expected to be less faithful to specific sites; and (3) majors should be observed in aggressive interactions more often. We individually marked all the individuals from a population of Longiperna during the reproductive season and recorded the location of each sighting for males and females as well as the identity of males involved in fights. Majors were more likely to have harems, and large majors were even more likely to do so. Majors were more philopatric and all males involved in fights belonged to this morph. These results strongly suggest that the mating tactic of the majors is based on resource defense whereas that of the minors probably relies on sneaking into the territories of the majors and furtively copulating with females.

Reproductive cycle of Ophionereis reticulata (Ophiuroidea, Echinodermata) on the southeast coast of Brazil

The reproductive cycle of Ophionereis reticulata, a common sediment-rocky shore-interface ophiuroid, was examined monthly from January 2002 to January 2003 at Praia Grande beach (Sao Sesbatiao, state of Sao Paulo, Brazil). Mature individuals were found from January 2002 to April 2002 and from November 2002 to January 2003. Spawning was regcorded from January 2002 to March 2002 and from November 2002 to January 2003. Mature sperm is still present in April which could be involved in the fertilization of the last oocytes spawned in March and April. November and December marked the final point in the maturation process, with a high concentration of yolk and lipid nutrients in the oocytes. Ophionereis reticulata showed, for the period analyzed, a single spawning period, during spring and summer.

Fluctuating abundance of humpback whales (Megaptera novaeangliae) in a calving ground off coastal Brazil

The humpback whale (Megaptera novaeangliae) population that uses Abrolhos Bank, off the east coast of Brazil as a breeding ground is increasing. To describe temporal changes in the relative abundance of humpback whales around Abrolhos, seven years (1998-2004) of whale count data were collected during July through to November. During one-hour-scans, observers determined group size within 9.3 km (5 n.m.) of a land-based observing station. A total Of 930 scans, comprising 7996 sightings of adults and 2044 calves were analysed using generalized linear models that included variables for time of day, day of the season, years and two-way interactions as possible predictors. The pattern observed was the gradual build-up and decline in whale counts within seasons. Patterns and peaks of adult and calf counts varied among years. Although fluctuation was observed, there was generally an increasing trend in adult counts among years. Calf counts increased only in 2004. These fluctuations may have been caused by some environmental conditions in humpback whales` summering grounds and also by changes in spatial-temporal concentrations in Abrolhos Bank. The general pattern observed within the study area mirrored what was observed in the whole Abrolhos Bank. Knowledge of the consistency with which humpback whales use this important nursing area should prove beneficial for designing future monitoring programmes especially related to whale watching activities around Abrolhos Archipelago.

Mating behavior of Sickius longibulbi (Araneae, Theraphosidae, Ischnocolinae), a spider that lacks spermathecae

We describe the mating behavior in the spermatheca-lacking theraphosid species Sickius longibulbi Soares & Camargo 1948. The behavior in captivity of nine pairs of S. longibulbi was videotaped and analyzed. The matting of this species presented an uncommon theraphosid pattern. There is little in the way of overt courtship by the male, the primary behavior seen being the male`s use of legs I and II to touch the female`s first pairs of legs and her chelicerae. Sometimes the male clasped one of the female`s first pairs of legs, bringing her close to him. While the female raised her body, the male clasped her fangs and held her tightly with his legs III wrapped around her prosoma. The male seemed to try to knock the female down, pushing her entire body until she lay on her dorsum. In this phase we observed the male biting the female on the sternum or on the leg joints. When the female fell, the male attempted to position himself at an angle of 90 degrees from the female. These movements appear to demand a lot of energy, particularly because the female is not passive during the mating. Our findings suggest that copulating in this position is, for the male, more successful than adopting other positions because it allows his extremely long palpal bulbs to deposit more sperm in the female oviduct where - since she lacks spermathecae - she retains the sperm. We suggest that the further he reaches into the oviduct, the greater the chance that he will fertilize the female`s eggs.