Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates

It is widely accepted that sperm morphology is a good indicator of fertility and it has been proposed that sperm quality may be related to subtle changes in sperm head morphology. However, a precise estimation of the morphology of ram sperm would be very useful to improve reproductive success in ovine. Computer-assisted morphometric analysis and clustering analysis have been important tools to study sperm subpopulations in domestic animals. However, to the best of our knowledge, no data exist studing morphometric differences regarding to sperm subpopulations within the ovine ejaculate. The aim of this study was to test the presence and distribution of sperm morphometric subpopulations in cryopreserved ejaculates from yearling and mature rams using an objective method by computer analysis system and to establish the relationship between the distribution of the subpopulations found and sperm quality in each individual ram. Principal component analysis revealed that three principal components for yearlings and four components for mature rams that represented more than 84% of the cumulative variance in both cases. After cluster analysis, three sperm morphometric subpopulations for yearlings (CLY) and four for mature (CLM) rams were identified with defined sperm dimensions and shapes. CLY1 included big, round and short sperm (37%), CLY2 included average size and slightly elliptical and elongated sperm (48%), CLY3 included small, long, elliptical and elongated sperm cells (15%). CLM1 consisted of average size and moderate elliptical and elongated (26%), CLM2 consisted of small, long, elliptical and elongated (31%), CLM3 consisted of small and round (32%) and CLM4 included big, short and round (8%) spermatozoa respectively. There were significant differences in the distribution of the three subpopulations (P < 0.001) as well as in the sperm concentration, total motility (%), sperm viability (%) and the overall (P < 0.05) in the ejaculates among the four yearling rams tested. Same results were found for the four subpopulations and the different sperm quality parameters in the ejaculates among the four mature rams tested. In conclusion, cryopreserved ram semen showed a specific structure with regard to sperm morphometric subpopulations. In addition, the distribution of these subpopulations seems to be related to stud maturity age and the ejaculate quality which would be a very important indicator of sperm function. Thus, analysis of sperm morphometric subpopulation structure together with functional tests could provide valuable information to assess the cryoresistence of ram spermatozoa.     

Male barn swallows use different signalling rules to produce ornamental tail feathers

The evolution of secondary sexual characters is the subject of controversial debate between those defending their role as ‘viability indicators’ and those arguing that ornaments are purely ‘attractive traits’ selected by females. Recent theoretical studies suggest that these hypotheses are not mutually exclusive, as both viability and attractiveness can contribute to improve the reproductive success of progeny and could thus simultaneously underlie female choices. If that is the case, strategies of cheaper advertisement, allowing the expression of larger ornaments for the same cost, could proliferate even in species in which honest signalling of viability prevails. Under this scenario, different males could invest a different amount of resources per ornament unit of expression, thus using different signalling rules. We studied the relationship between tail feather length (a trait that is the subject of a female mate preference) and feather mass (a measure of investment in feather production) in a barn swallow Hirundo rustica population. Different males used different and consistent signalling rules when developing ornamental feathers. That is, to produce a feather of a given length, each male used a constant amount of resources across different years, but this amount varied between males. Although the amount of material invested in feathers (feather mass) is a condition-dependent trait, the organization of this material in ornamental feathers (i.e. the signalling rules) was not. Neither survival nor risk of feather breakage was related to the signalling rules. Thus, these results suggest that both ‘viability’ and ‘runaway’ mechanisms are independent determinants of the evolution of ornamental sexual feathers in the barn swallow. A preference for long tails will ensure that females either obtain a sire with high viability, or one transferring the capability to produce longer and more attractive tails at a lower cost of production to its offspring.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

A Phase I,Dose-Finding Study of Sorafenib in Combination with Gemcitabine and Radiation Therapy in Patients with Unresectable Pancreatic Adenocarcinoma: A Grupo Espa?ol Multidisciplinario en Cáncer Digestivo (GEMCAD) Study

Purpose

Sorafenib, an oral inhibitor of B-raf, VEGFR2, and PDGFR2-beta, acts against pancreatic cancer in preclinical models. Due to the radio-sensitization activity of both sorafenib and gemcitabine, we designed a multicenter, phase I trial to evaluate the safety profile and the recommended dose of this combination used with concomitant radiation therapy.

Methods

Patients with biopsy-proven, unresectable pancreatic adenocarcinoma (based on vascular invasion detected by computed tomography) were treated with gemcitabine (300 mg/m2 i.v. weekly ×5 weeks) concurrently with radiation therapy (45 Gy in 25 fractions) and sorafenib (escalated doses in a 3+3 design, from 200 to 800 mg/day). Radiation portals included the primary tumor but not the regional lymph nodes. Patients with planning target volumes (PTV) over 500 cc were excluded. Cases not progressing during chemoradiation were allowed to continue with sorafenib until disease progression.

Results

Twelve patients were included. Three patients received 200 mg/day, 6 received 400 mg/day, and 3 received 800 mg/day; PTVs ranged from 105 to 500 cc. No dose-limiting toxicities occurred. The most common grade 2 toxicities were fatigue, neutropenia, nausea, and raised serum transaminases. Treatment was discontinued in one patient because of a reversible posterior leukoencephalopathy. There were no treatment-related deaths.

Conclusion

The addition of sorafenib to concurrent gemcitabine and radiation therapy showed a favorable safety profile in unresectable pancreatic adenocarcinoma. A dose of 800 mg/day is recommended for phase II evaluation.

Trial Registration

EudraCT 2007-003211-31 ClinicalTrials.gov 00789763     

Rif1 Regulates Initiation Timing of Late Replication Origins throughout the S. cerevisiae Genome

Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4Δ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1Δ cells, and checkpoint-defective mec1Δ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres.     

Decline of Native Freshwater Fishes in a Mediterranean Watershed on the Iberian Peninsula: A Quantitative Assessment

We examined changes in the distribution of 9 native and 18 introduced freshwater fishes in the south-eastern Pyrenees watershed, Iberian Peninsula, using data from 1996, 1984–1988 and historical information. This region suffers many modifications to its freshwater ecosystems that are linked to human activity in the Mediterranean regions. Fish communities, stream physical habitat and environmental degradation were assessed at 168 sites from 11 basins in 1996. Seven native species (78%) showed decline from previous data, one of which became extirpated in the first half of the 20th century. On the other hand, introduced species are expanding. As a consequence, intact native communities are increasingly rare, declining from presence in 22% of river courses in 1984–1988 to 15% in 1996. The most typical community type is a mixture of native and introduced species occupying 30% of river courses. Stream degradation seems to be the main cause of this process because fish communities differed between degraded streams and streams suffering less impact. A principal component analysis showed that water pollution and modifications to the habitat were the two anthropogenic factors that accounted for most changes in the fish community integrity. Habitat alteration, primarily through construction of dams and water diversions, has fragmented habitats and isolated native fish communities in headwater streams. Current protection measures do not offer effective conservation of threatened species and communities. A global conservation and restoration programme from an ecosystem-based approach is essential to reverse the trend affecting native freshwater fishes in this Mediterranean region.     

Spectral Dependence of Photoregulation of Inorganic Nitrogen Metabolism in Chlamydomonas reinhardii

The utilization of NO₃⁻ by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of NO₂⁻ and NH₄⁺ to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The stimulation of NO₃⁻ utilization and of its associated NO₂⁻ and NH₄⁺ excretions saturated at very low quantum fluxes of blue light (15 microequivalents per square meter per second) in Chlamydomonas reinhardii cells sparged with CO₂-free air and irradiated with 50 microequivalents per square meter per second background red light. The wavelength dependence data of this stimulation correlated closely with the in situ photoactivation of nitrate reductase and also with the light induced increase in its biosynthesis and/or assembly.

These results indicate that the photoregulation of inorganic N metabolism in C. reinhardii is mainly due to the blue light modulation of nitrate reductase. Although flavins are the most suitable candidates to act as physiological photoreceptors, the wavelength dependence data only show a major peak in the blue region between 400 and 500 nanometers.

     

MARCKS phosphorylation by PKC strongly impairs cell polarity in the chick neural plate

Neurulation involves a complex coordination of cellular movements that are in great part based on the modulation of the actin cytoskeleton. MARCKS, an F‐actin‐binding protein and the major substrate for PKC, is necessary for gastrulation and neurulation morphogenetic movements in mice, frogs, and fish. We previously showed that this protein accumulates at the apical region of the closing neural plate in chick embryos, and here further explore its role in this process and how it is regulated by PKC phosphorylation. PKC activation by PMA caused extensive neural tube closure defects in cultured chick embryos, together with MARCKS phosphorylation and redistribution to the cytoplasm. This was concomitant with an evident disruption of neural plate cell polarity and extensive apical cell extrusion. This effect was not due to actomyosin hypercontractility, but it was reproduced upon MARCKS knockdown. Interestingly, the overexpression of a nonphosphorylatable form of MARCKS was able to revert the cellular defects observed in the neural plate after PKC activation. Altogether, these results suggest that MARCKS function during neurulation would be to maintain neuroepithelial polarity through the stabilization of subapical F‐actin, a function that appears to be counteracted by PKC activation.