Ultrastuctural and cytochemical study of spermatogenesis in Scrobicularia plana (Mollusca,Bivalvia)

The ultrastructure of the mature spermatozoa and spermatogenesis of the bivalve Scrobicularia plana are described. Support cells extend from the basal lamina to the lumen of the testis and are laterally connected to the germinal epithelium. Germ cells present intercellular bridges and flagella since the spermatogonial stage. While spermatogonia and spermatocytes appear connected to support cells by desmosome-like junctions, elongated spermatids are held at the acrosomal region by support cell finger-like processes. During spermiogenesis, the acrosomal vesicle differentiates from a golgian saccule and then migrates to the nuclear apex. A microtubular manchette arising from centrioles surrounds the acrosomal vesicle, the nucleus, and the mitochondria at the time these three organelles start their elongation, disappearing after that. The mature spermatozoon of S. plana lacks a distinct midpiece because the mitochondria extend from the region of the pericentriolar complex along the nucleus anteriorly for approximately 1.4 μm. The features of this bivalve type of modified spermatozoon are compared with those of other animal groups having similar modifications.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

An unethical trial and the politicization of the COVID-19 pandemic in Brazil: The case of Prevent Senior

The Brazilian Federal Senate created a Parliamentary Inquiry Commission (CPI) to investigate the Bolsonaro government's irregularities in the management of the COVID-19 pandemic. One of the cases that drew attention was the research conducted by Prevent Senior, a private health insurance company, on the early treatment of COVID-19. The article analyzes the scientific validity of the research and the ethical problems related to its implementation. It is based on analysis of Prevent Senior's report of the clinical study, the Brazilian and USA clinical trial registries, the Senate's CPI report, and on the information reported by the media. This case of scientific fraud and political-ideological bias exemplifies how Prevent Senior, using a questionable protocol to enhance its reputation and gain government support, was instrumental in building the “early treatment” narrative for COVID-19, and shows how it served as a basis for a government public policy that promoted the use of ineffective drugs.     

大河岸线生态完整性内涵及评估方法——以长江岸线为例

大河岸线作为流域的重要生态空间,其内涵属性、空间格局和生态功能都具有显著的整体性。从岸线的陆域原真性、水陆生态渐变性、生境依存性及生物多样性等维度,分析了大河岸线生态完整性的科学内涵。结合国内外河流岸线生态评价研究,建立了大河岸线生态完整性的科学表征体系,提出各项指标评估和综合分级方法,并对长江岸线生态完整性进行了评估。结果表明,长江岸线生态完整性总体处于中等水平,从上游到下游沿岸各地市岸段生态完整性得分总体逐步降低;中上游地市岸段受到滨江湿地和洲岛发育程度及湿地保护状况等因素制约,下游地市岸段则受土地开发强度、自然岸线保有率、水陆连通性等制约。虽然中下游岸线有较高的开发程度且受长江大堤的约束,生态完整性受到明显影响,但在相同条件下生态完整性仍然有较大差异性,部分地区如铜陵段、安庆段和上海段仍保持了较高的完整性。大河岸线生态完整性的评估有助于形成既尊重自然规律、保护生态环境、保障防洪安全及供水安全,又能支撑社会经济发展、促进高效集约利用的长江生态岸线格局。     

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.     

Animal board invited review: An update on the methods for semen quality evaluation in swine – from farm to the lab

Pig breeding is mainly conducted through artificial insemination with liquid-stored semen. It is, therefore, crucial to ensure that sperm quality is over the standard thresholds, as reduced sperm motility, morphology or plasma membrane integrity are associated with reduced farrowing rates and litter sizes. This work aims to summarise the methods utilised in farms and research laboratories to evaluate sperm quality in pigs. The conventional spermiogram consists in the assessment of sperm concentration, motility and morphology, which are the most estimated variables in farms. Yet, while the determination of these sperm parameters is enough for farms to prepare seminal doses, other tests, usually carried out in specialised laboratories, may be required when boar studs exhibit a decreased reproductive performance. These methods include the evaluation of functional sperm parameters, such as plasma membrane integrity and fluidity, intracellular levels of calcium and reactive oxygen species, mitochondrial activity, and acrosome integrity, using fluorescent probes and flow cytometry. Furthermore, sperm chromatin condensation and DNA integrity, despite not being routinely assessed, may also help determine the causes of reduced fertilising capacity. Sperm DNA integrity can be evaluated through direct (Comet, transferase deoxynucleotide nick end labelling (TUNEL) and its in situ nick variant) or indirect tests (Sperm Chromatin Structure Assay, Sperm Chromatin Dispersion Test), whereas chromatin condensation can be determined with Chromomycin A3. Considering the high degree of chromatin packaging in pig sperm, which only have protamine 1, growing evidence suggests that complete decondensation of that chromatin is needed before DNA fragmentation through TUNEL or Comet can be examined.     

A qualitative procedure for the assessment of the habitat integrity status of the Luvuvhu River (Limpopo system,South Africa)

The riparian zone and instream habitat integrity of the Luvuvhu River were assessed based on a qualitative rating of the impacts of major disturbance factors such as water abstraction, flow regulation, bed and channel modification, etc. A system was devised to assess the impact of these factors on the relative frequency and variability of habitats on a spatial and temporal scale gauged against habitat characteristics that could have been expected to occur under conditions not anthropogenically influenced. It was found that deterioration of habitat integrity can be ascribed primarily to water abstraction. This has resulted in the cessation of surface flow in a naturally perennial river during the dry season and during droughts with consequent tree deaths and a loss of fast flowing instream habitat types in the main stem of the river. The relatively small high rainfall area in the catchment, the highly variable rainfall pattern and the occurrence of sporadic severe droughts exacerbate the impact of water abstraction on the instream and riparian habitats with expected detrimental consequences for the associated biota. The effect of water abstraction is particularly severe in the lower part of the river which flows through the Kruger National Park as no perennial tributaries join the Luvuvhu River in this section. Other factors which affect the habitat integrity of the river are the removal of indigenous riparian vegetation in some river sections, encroachment by exotic vegetation, bank erosion and stream bed modification.     

Signal transduction pathways in guinea pig sperm

Trifluoperazine (TFP), the antagonist of calmodulin (CaM). significantly stimulated the capacitation and acrosome reaction of guinea pig spermatozoa at the concentration of 10-100μmol/L, independent of the external Ca2+. Forskolin, dbcAMP and caffeine evidently promoted the occurrence of acrosome reaction of spermatozoa at early capacitation stage (5 h) in nonsynchronous system but not in synchronous system. If the spermatozoa were capacitated for 15 h in synchronous system, the above three drugs significantly stimulated acrosome reaction in a Ca2+-independent manner. Protein kinase C activators, i.e. phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB) did not influence the occurrence of acrosome reaction of spermatozoa at early capacitation stage, but significantly increased the acrosome reaction rate in capacitated spermatozoa in a Ca2+-independent manner. In contrast. PKC inhibitor staurosporine significantly inhibited the occurrence of acrosome reaction.     

Conservation and management issues of Prespa National Park

The present article describes the human activities inthe area and their changes and trends, given that thecontemporary nature conservation key issues areclosely related to past and present socio-economic andcultural conditions, both within Prespa itself andoutside of it. The liberation of Prespa, a remote andrather socially isolated borderline area, from Ottoman occupation took place in 1912–13. Recentevents in its environmental history have been forestclearings and human depopulation in the Civil War(1944–49), irrigation system construction in the1960s, its discovery by ornithologists in the late1960s, the high emigration rates of 1960–1980, itsdesignation as a National Park in 1974, thedestructive development works of 1984–86 afterGreeces entry to the EEC and the conversion to intensivebean cultivation in the mid 1980s.The human population of ca 7000 at the start of thiscentury suffered an almost 80% decrease after theCivil War. A resettlement in the mid 1950s increasedthe population by 40% but in the 1970s emigrationreduced it to its previous size of around 1500, stillits present size. The extensive and diverse farmingsystems of previous centuries changed gradually afterthe construction of an irrigation network in the1960s, which twenty years later and along withinternational changes in production and economypatterns, made possible the present day domination ofa bean monoculture within irrigated croplands. Thiswas accompanied by increased energy inputs,mechanisation and the use of chemicals.Intensification of agriculture brought increasedincomes which attracted also livestock keepers,resulting in a 56% decrease in numbers of livestockin 1964–1993. Cattle are presently kept for meatproduction only. They graze on the mountains, and nolonger in the meadows around the lakeshore. A localshorthorn breed accounting for 93% of all cattle in1963 declined to less than 18% in 1993. Fishing aimedmainly at carp and Prespa bleak, continues today as inthe past to be a source of supplementary income.Eighty-six per cent of the – mainly oak and beech –forests are State owned and the rest belong to localmunicipalities. The forest area has not changedessentially in the last 40 years, but forest roadshave expanded. Forests provide mainly fuelwood forlocal needs and small quantities of industrial timber.No specific management is conducted to safeguard theconservation values of the forest, the majority ofwhich consists of even aged coppiced stands withlimited value for wildlife. Manufacturing never playedan important role for the economy of the area. Tourismhas increased in the last 20 years, concentratedmainly in summer and spring. Accommodation in thevillages is not yet satisfactory though it hasimproved enormously in recent years. The NationalParks infrastructure for visitors remains poor.Tourist pressure generally is not yet that high toimpact drastically the social and environmentalfabric. In places, hunting, angling and poaching cannegatively affect the target species. The constructionof the irrigation network in the eastern part of LakeMikri Prespa was the most important change to thelandscape and the habitats of Prespa the last 30years. It resulted in the embankment of free-flowingstreams, drainage of wet meadows and dramaticreduction of trees and hedges. In the last 15 years,the reedbeds, no longer burnt or mowed and assisted byincreased nutrient loadings from agricultural runoff,have expanded landward to occupy formerly open,periodically flooded, areas. The above changes inhabitats and land use have brought an impoverishmentof biodiversity which can be seen in the exterminationor the decrease in the numbers of certain plant andbird species.The problems preventing a better management of theNational Park and the perpetuation of its values areinappropriate legislation, the poor degree ofcoordination of the public services, the lack of botha specific National Park Service in Greece and amanagement authority for the Park and the hostileattitude of local people due to their limitedinformation and involvement. The scenic beauty, thewetlands and a number of rare habitats, the diversityof habitats, flora and fauna, the endemic life forms,the rare mammals and the colonial waterbirds, thelocal architecture and the cultural expressions suchas the Byzantine monuments, are the values of Prespathat must be preserved as a whole. The initialconservation efforts aimed at preserving biodiversitybut later it became apparent that not only are humansan inseparable part of the whole, but additionally,several of their extensive activities have contributedsubstantially to the rich biodiversity of the area.Through the prerequisites of keeping a balance betweenprimary and tertiary sector activities and ofrevitalizing the social fabric, the following are thekey management and conservation issues of today: anunequivocal agreement on the values to be preservedand their limits; the formation of a managementauthority; a new realistic protection and land usezoning; the diversification and extensification of allfarming activities; the restoration of the wet meadowhabitats; the ensuring of a high water level and agood water quality for both lakes through tri-lateralcooperation between Greece, Albania and FYROM; and theestablishment of a permanent environmental monitoringscheme.     

Effects of calcium,magnesium, potassium and boron on sperm cells isolated from pollen of Zea mays L.

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not appropriate for the maintenance of isolated maize (Zea mays L.) sperm cells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hemacytometry. At 0.01 and 0.1 mM, there were no differences in cell number between control and any salt-treated cells except a 22% decrease with 0.1 mM MgSO₄ at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO₃)₂ and MgSO₄, with loss of integrity of most cells at 48 h, while KNO₃ and H₃BO₃ had little or no effect. Further characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free calcium by addition of EGTA to the suspension medium improved cell longevity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12% of cells remained intact at 24 h. The reduction in cell integrity was hastened when cells were pretreated with the calcium ionophore A23187 prior to exposure to 1 mM calcium, with a 54% reduction in cell number at 1 h and complete cell lysis at 24 h. However, depletion of cytosolic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid reduction of cell integrity as well. These results collectively suggest that maize sperm cells are sensitive to exogenous free calcium; however, a certain level of cytosolic free calcium is necessary for maintenance of integrity. Mechanisms of calcium-induced reduction in cell integrity are discussed along with possible roles of the sensitivity of sperm cells to calcium in fertilization.