Proteomic characteristics of human sperm cryopreservation

Human sperm cryopreservation in assisted reproductive technology is the only proven method that enables infertile men to father their own children. However, freezing and thawing reduces spermatozoon motility, viability, and fertilizing ability. An association between dysfunctional spermatozoa due to cryoinjury and protein changes has not been established. We investigated through proteomic analysis the differential protein characteristics between freeze‐thawed and fresh sperm samples obtained from nine normozoospermic donors. Twenty‐seven proteins differed in abundance between the two groups, and results were verified for four proteins via Western blot and immunofluorescent staining. These proteins are putatively involved in sperm motility, viability, acrosomal integrity, ATP and isocitrate content, mitochondrial membrane potential, capacitation, acrosome reaction, and intracellular calcium concentration. These marked differences suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.     

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing–thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/W, circularity: 4πA/P², elongation: (L − W)/(L + W), regularity: πLW/4A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area (r = 0.75 and r = 0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter (r = 0.65 and r = 0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.     

Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda)

Levron, C., Yoneva, A. and Kalbe, M. 2011. Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda). —Acta Zoologica (Stockholm) 00 : 1–8. The spermiogenesis and the mature spermatozoon of Schistocephalus solidus (Cestoda: Diphyllobothriidea) are described using transmission electron microscopy. Spermiogenesis in S. solidus begins with the formation in the spermatid of a differentiation zone surrounded by cortical microtubules and delimited by arching membranes. This conical area presents two centrioles associated with striated rootlets and a median cytoplasmic extension between them. The centrioles are separated by an intercentriolar body composed of three electron‐dense plates dividing four electron‐lucent plates. The centrioles give rise to two flagella that undergo a rotation and later fuse proximodistally with the median cytoplasmic expansion. The presence of an electron‐dense material in the distal part of the differentiation zone is observed in the early stage of spermiogenesis. This pattern corresponds to Type I spermiogenesis according to the classification proposed by Bâ and Marchand (Mémoires du Muséum National d’Histoire Naturelle 1995; 166 : 87). The mature spermatozoon of S. solidus presents the Type I pattern defined by Levron et al. (Biological Reviews 2010; 85 : 523). It consists of five regions that exhibit two axonemes, parallel cortical microtubules, nucleus and electron‐dense zones. The anterior tip of the spermatozoon possesses only a few singlets. The axonemes are of a 9 + ’1’ trepaxonematan pattern and do not reach the posterior extremity of the mature spermatozoon.     

Prmt5: a guardian of the germline protects future generations

Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage that form sperm and egg cells. It is of great importance to preserve the germline from DNA damage and potentially from epimutations in order to ensure the survival of future generations. Recent research highlights the role of the protein arginine methyltransferase 5 (PRMT5) as an important player in DNA protection during germline development in the mouse (Kim et al, 2014 & Li et al, 2015 ).     

Japanese eel (Anguilla japonica Temminck & Schlegel, 1846) propagation using cryopreserved sperm samples

The aim of this study was to test the artificial seminal plasma (ASP) as the extender as well as methanol as the cryoprotectant for cryopreservation, and to collect information on the fertilizing capacity of cryopreserved sperm samples. Eggs from Japanese eel, Anguilla japonica, females were control‐fertilized with native sperm diluted with ASP and cryopreserved sperm (ASP as extender [in 1:100 ratio] and 10% methanol in v/v final concentration). No statistical differences (p < .05) were among the measured parameters (hatching, survival after 10 days post‐hatch, malformation rates) between the two groups, thus this cryopreservation method can be used effectively for artificial propagation of the Japanese eel, Anguilla japonica.     

Digest: Unlocking the creative potential of specialization*

Do constant environments produce ecological specialists? Wang et al. test this common assumption in Drosophila melanogaster and find that the converse may be true: constant environments sometimes produce robust generalists that can withstand change. In this study, increased tolerance to change may be best explained as a by‐product of adaptation to a particularly harsh salt‐enriched environment.     

Distance from forest edge affects bee pollinators in oilseed rape fields

Wild pollinators have been shown to enhance the pollination of Brassica napus (oilseed rape) and thus increase its market value. Several studies have previously shown that pollination services are greater in crops adjoining forest patches or other seminatural habitats than in crops completely surrounded by other crops. In this study, we investigated the specific importance of forest edges in providing potential pollinators in B. napus fields in two areas in France. Bees were caught with yellow pan traps at increasing distances from both warm and cold forest edges into B. napus fields during the blooming period. A total of 4594 individual bees, representing six families and 83 taxa, were collected. We found that both bee abundance and taxa richness were negatively affected by the distance from forest edge. However, responses varied between bee groups and edge orientations. The ITD (Inter‐Tegular distance) of the species, a good proxy for bee foraging range, seems to limit how far the bees can travel from the forest edge. We found a greater abundance of cuckoo bees (Nomada spp.) of Andrena spp. and Andrena spp. males at forest edges, which we assume indicate suitable nesting sites, or at least mating sites, for some abundant Andrena species and their parasites (Fig.  1 ). Synthesis and Applications. This study provides one of the first examples in temperate ecosystems of how forest edges may actually act as a reservoir of potential pollinators and directly benefit agricultural crops by providing nesting or mating sites for important early spring pollinators. Policy‐makers and land managers should take forest edges into account and encourage their protection in the agricultural matrix to promote wild bees and their pollination services.

Figure 1 Open in figure viewer PowerPoint Left, a Nomada sp male; right, an Andrena sp male. Caption Left, a Nomada sp male; right, an Andrena sp male.

Introduction

Pollinators play an important functional role in most terrestrial ecosystems and provide a key ecosystem service (Ashman et al. 2004 ). Insects, particularly bees, are the primary pollinators for the majority of the world's angiosperms (Ollerton et al. 2012 ). Without this service, many interconnected species and processes functioning within both wild and agricultural ecosystems could collapse (Kearns et al. 1998 ). Brassica napus (oilseed rape, OSR) represents the most widespread entomophilous crop in France with almost 1.5 Mha in 2010 (FAOSTAT August 10th, 2012). Results differ between varieties, but even though it seems that OSR produces 70% of its fruits through self‐pollination (Downey et al. 1970 in Mesquida and Renard 1981 ), native bees are also known to contribute to its pollination (Morandin and Winston 2005 ; Jauker et al. 2012 ). Bee pollination leads to improved yields (Steffan‐Dewenter 2003b ; Sabbahi et al. 2005 ) and to a shorter blooming period (Sabbahi et al. 2006 ), thus increasing the crop's market value (Bommarco et al. 2012 ). The most widely used species in crop pollination is the honeybee (Apis mellifera L) which is sometimes assumed to be sufficient for worldwide crop pollination (Aebi and Neumann 2011 ). However, this assertion has been questioned by different authors (Ollerton et al. 2012 ), and several studies show that many wild bees are also efficient pollinators of crops (Klein et al. 2007 ; Winfree et al. 2008 ; Breeze et al. 2011 ). Recently, Garibaldi et al. ( 2013 ) found positive associations of fruit set with wild‐insect visits to flowers in 41 crop systems worldwide. They demonstrate that honeybees do not maximize pollination, nor can they fully replace the contributions of diverse, wild‐insect assemblages to fruit set for a broad range of crops and agricultural practices on all continents with farmland. Unfortunately, not only are honey bees declining due to a variety of different causes (vanEngelsdorp et al. 2009 ), wild bee populations are also dwindling (Potts et al. 2010 ). Their decline has been documented in two Western European countries (Britain and the Netherlands) by comparing data obtained before and after 1980 (Biesmeijer et al. 2006 ). These losses have mostly been attributed to the use of agrochemicals, the increase in monocultures, the loss of seminatural habitat and deforestation (Steffan‐Dewenter et al. 2002 ; Steffan‐Dewenter and Westphal 2008 ; Brittain and Potts 2011 ). Several studies have shown the importance of natural or seminatural habitats in sustaining pollinator populations or pollination services close to fruit crops (Steffan‐Dewenter 2003a ; Kremen et al. 2004 ; Greenleaf and Kremen 2006a ; Carvalheiro et al. 2010 ). Morandin and Winston ( 2006 ) presented a cost–benefit model that estimates profit in OSR agroecosystems with different proportions of uncultivated land. They calculated that yield and profit could be maximized with 30% of the land left uncultivated within 750 m of field edges. Other studies have demonstrated a negative impact of the distance from forests on pollination services or bee abundance and richness both in tropical ecosystems (De Marco and Coelho 2004 ; Blanche et al. 2006 ; Chacoff and Aizen 2006 ) and in temperate ecosystems (Hawkins 1965 ; Taki et al. 2007 ; Arthur et al. 2010 ; Watson et al. 2011 ). These studies all suggest that natural or seminatural habitats are important sources of pollinators, probably because they provide “partial habitats” (Westrich 1996 ) such as complementary mating, foraging, nesting, and nesting materials sites that bees need to complete their life cycle. In this study, we focused on the effect of distance to forest edge on bee assemblages in OSR ecosystems. Forest edges could provide one or more important partial habitats for different bee species in agricultural landscapes, in particular when associated with a mass‐flowering crop such as OSR (Le Feon et al. 2011 ). For example, the availability of untilled soil and dead branches might provide ground‐nesting and cavity‐nesting bee species with numerous nesting sites. Moreover, during spring at least, the understory and the forest edge can provide cover containing flowering plants and wild trees such as Prunus spp, Castanea sativa, or Salix spp and thereby allow bees to find alternative floral resources. During spring 2010 and 2011, in two areas in France, we examined wild bee abundance and taxa richness both along forest edges and inside OSR fields at different distances from the forest. Like other taxa, bees respond to environmental variables according to their biologic traits that determine access and requirements for nesting, mating, and forage resources, species mobility or physiological tolerance. Specifically, we hypothesized that (1) bee abundance, species richness, and composition of bee communities within the crop field are dependent on the distance from the forest edge (where complementary floral resources, nesting sites, shelters, etc. can be found) and on the orientation of the forest edge; (2) the identity of bees in the crop is related to their foraging range which we measured with the ITD (Inter‐Tegular distance); (3) the forest edge may be the nesting or mating sites for cavity‐nesting or ground‐nesting bees such as Osmia spp or Andrena spp which are important groups of potential early spring pollinators for OSR.     

Mutually exclusive phylogenomic inferences at the root of the angiosperms: Amborella is supported as sister and Observed Variability is biased

Identifying the extant sister group to the remaining angiosperms has been a subject of long debate, for which the primary currently competing hypotheses are that Amborella alone is sister or that the clade (Amborella, Nymphaeales) is sister. Both Xi et al. (Syst. Biol., 2014, 63, 919) and Goremykin et al. (Syst. Biol., 2015, 64, 879) identified Amborella as sister in concatenation‐based phylogenetic analyses of their 310 nuclear genes and 78 plastid genes, respectively. But after application of Observed Variability‐based character subsampling, both papers reported the clade (Amborella, Nymphaeales) as sister. Hence alternative character‐sampling strategies may produce highly supported yet mutually exclusive phylogenetic inferences when applied to nuclear and plastid genomic data sets. Edwards et al. (Mol. Phylogenet. Evol., 2016, 94, 447) defended Observed Variability and the (Amborella, Nymphaeales) hypothesis. In this study I respond to Edwards et al.'s (Mol. Phylogenet. Evol., 2016, 94, 447) criticisms of Simmons and Gatesy (Mol. Phylogenet. Evol., 2015, 91, 98) and use Edwards et al.'s (Mol. Phylogenet. Evol., 2016, 94, 447) and Goremykin et al.'s (Syst. Biol., 2015, 64, 879) own data to demonstrate that the best‐supported phylogenetic hypothesis is that Amborella alone is sister and that the competing evidence in favour of the (Amborella, Nymphaeales) hypothesis is caused primarily by methodological artifacts (biased character deletion by Observed Variability, MP‐EST and STAR generally not being robust to the highly divergent and mis‐rooted gene trees that were used).     

Demystifying the RAD fad

We are writing in response to the population and phylogenomics meeting review by Andrews & Luikart ( 2014 ) entitled ‘Recent novel approaches for population genomics data analysis’. Restriction‐site‐associated DNA (RAD) sequencing has become a powerful and useful approach in molecular ecology, with several different published methods now available to molecular ecologists, none of which can be considered the best option in all situations. A&L report that the original RAD protocol of Miller et al. ( 2007 ) and Baird et al. ( 2008 ) is superior to all other RAD variants because putative PCR duplicates can be identified (see Baxter et al. 2011 ), thereby reducing the impact of PCR artefacts on allele frequency estimates (Andrews & Luikart 2014 ). In response, we (i) challenge the assertion that the original RAD protocol minimizes the impact of PCR artefacts relative to that of other RAD protocols, (ii) present additional biases in RADseq that are at least as important as PCR artefacts in selecting a RAD protocol and (iii) highlight the strengths and weaknesses of four different approaches to RADseq which are a representative sample of all RAD variants: the original RAD protocol (mbRAD, Miller et al. 2007 ; Baird et al. 2008 ), double digest RAD (ddRAD, Peterson et al. 2012 ), ezRAD (Toonen et al. 2013 ) and 2bRAD (Wang et al. 2012 ). With an understanding of the strengths and weaknesses of different RAD protocols, researchers can make a more informed decision when selecting a RAD protocol.     

Exploiting plants for glutathione (GSH) production: Uncoupling GSH synthesis from cellular controls results in unprecedented GSH accumulation

Glutathione (GSH) is a key factor for cellular redox homeostasis and tolerance against abiotic and biotic stress ( May et al., 1998 ; Noctor et al., 1998a ). Previous attempts to increase GSH content in plants have met with moderate success ( Rennenberg et al., 2007 ), largely because of tight and multilevel control of its biosynthesis ( Rausch et al., 2007 ). Here, we report the in planta expression of the bifunctional γ‐glutamylcysteine ligase—glutathione synthetase enzyme from Streptococcus thermophilus (StGCL‐GS), which is shown to be neither redox‐regulated nor sensitive to feedback inhibition by GSH. Transgenic tobacco plants expressing StGCL‐GS under control of a constitutive promoter reveal an extreme accumulation of GSH in their leaves (up to 12 μmol GSH/gFW, depending on the developmental stage), which is more than 20‐ to 30‐fold above the levels observed in wild‐type (wt) plants and which can be even further increased by additional sulphate fertilization. Surprisingly, this dramatically increased GSH production has no impact on plant growth while enhancing plant tolerance to abiotic stress. Furthermore, StGCL‐GS‐expressing plants are a novel, cost‐saving source for GSH production, being competitive with current yeast‐based systems ( Li et al., 2004 ).     

Assessing community and ecosystem sensitivity to climate change – toward a more comparative approach

Plant communities can vary widely in their sensitivity to changing precipitation regimes, as reported by Byrne et al., Mulhouse et al. and Sternberg et al. in this issue of Journal of Vegetation Science. But to understand why communities differ in their sensitivity, we argue that clearly defined metrics of sensitivity and coordinated research approaches are needed to elucidate mechanisms.     

Deciphering the biochemistry and identifying biomarkers to multiple sclerosis

Multiple sclerosis is an idiopathic demyelinating disease of the CNS. Despite being extensively studied during the last decades, many molecular aspects of the disease are still to be elucidated. Moreover, biomarkers for treatment and early diagnosis are major issues to be tackled. In this edition of Kroksveen et al. (Proteomics 2015, 15, 3361–3369) present biomarker candidates for the early detection of multiple sclerosis. Despite the need for validation in larger sets of samples, this dataset contributes to resolve open questions associated to multiple sclerosis.     

First beetle elytra,abdomen (Coleoptera) and a mine trace from Lunz (Carnian,Late Triassic,Lunz‐am‐See,Austria) and their taphonomical and evolutionary aspects

Abstract: Four new species of Permosynidae are described: Platycrossus caroli Ponomarenko in Meller et al. sp. nov., Hydrobiites handlirschi Ponomarenko in Meller et al. sp. nov., Ademosyne polyzetete Ponomarenko in Meller et al. sp. nov. and Diarcuipenna bennettitophila Ponomarenko in Meller et al. sp. nov. Together with one polyphagous abdomen, they represent the first beetle remains from the Lunz formation, known for its richness in bennettite and cycad leaf remains and also for the bennettite reproductive organs. Furthermore, the first insect mine trace on a Nilssonia leaf segment is described, which is one of the most ancient linear mine traces. The rareness of insect remains in the plant‐bearing layers of the Lunz formation is still an enigma from the taphonomical–chemical point of view. The palaeoentomological and palaeobotanical considerations tentatively indicate a relationship between the rare occurrence of coniferous plants and the evolution or radiation of phytophagous insects during this time.     

A clarification of Pouydebat et al., 2008, evolution of grasping among anthropoids

Several statements by Pouydebat et al. (2008) do not adequately represent views of authors cited, in part because they reflect confusion in the literature about terminology regarding precision gripping. We address these problems, by tracing definitions of precision grips through the literature on manipulative behaviour and identifying the grip that is central to the Pouydebat et al. (2008) study. This allows us to offer a clarification of the statements by Pouydebat et al. (2008) regarding the sequence of appearance of human grip capabilities and possible morphological correlates to these capabilities in extant species.     

The ultrastructural characters of the mature spermatozoon of Opechona bacillaris (Molin, 1859) (Digenea,Lepocreadiidae) a parasite of Scomber colias Gmelin, 1789 (Scombridae) off the coast of Dakar (Senegal)

This study presents the ultrastructure of the mature spermatozoon of Opechona bacillaris, a digenean belonging to the family Lepocreadiidae. The sperm cell of O. bacillaris exhibits the general pattern described in most of the Lepocreadioidea: two axonemes of the 9 + ‘1’ pattern of the Trepaxonemata, mitochondria, a cortical mitochondrion, a nucleus, electron‐dense material in the anterior extremity of the spermatozoon, external ornamentation of the plasma membrane with associated spinelike bodies, and granules of glycogen. However, particularities of O. bacillaris are the simultaneous presence in the anterior extremity of the spermatozoon of the electron‐dense material, a mitochondrion, and the absence of cortical microtubules. In the Lepocreadiidae, we describe for the first time in O. bacillaris spinelike bodies associated with the external ornamentation of the plasma membrane and two mitochondria. The first mitochondrion is moniliform and composed of a mitochondrial cord with joined mitochondrial bulges. The second mitochondrion shows a regular form. The posterior tip of the spermatozoon has only singlets to owing to the disorganization of the second axoneme and granules of glycogen as occurs in Hypocreadium caputvadum, the other studied species of the family Lepocreadiidae.     

High dose refuge strategies and genetically modified crops – reply to Tabashnik et al.

Cultivating non‐toxic conventional crops (refuges) in the proximity to transgenic crops that produce Bacillus thuringienesis (Bt) toxins is widely recommended to delay pest adaptation to these toxins. Using a spatially structured model of resistance evolution, Vacher and co‐workers (Vacher, C., Bourguet, D., Rousset, F., Chevillon, C. & Hochberg, M.E. 2003. J. Evol. Biol. 16 : 378–387.) show that the percentage of refuge fields required for the sustainable control of pests can be reduced through intermediate levels of refuge field aggregation and by lowering the toxin dose produced by Bt plants. Tabashnik, B.E., Gould, F. & Carrière, Y. (2004 J. Evol. Biol doi: 10.1111/j1420–9101.2004.00695.x) call into question the results of Vacher et al. (2003) concerning the effect of toxin dose. They argue that these results arise from invalid assumptions about larval concentration–mortality responses for the insect considered, the cotton pest Heliothis virescens. We show here that the models presented by Vacher et al. (2003) and Tabashnik et al. (2004) both show inaccuracies in their definitions of genotypic fitness. The level of dominance estimated by Tabashnik et al. (2004) from larval mortality rates data is irrelevant to resistance evolution, and the fitness cost of resistance evolution, and the fitness cost of resistance is inaccurately integrated into their framework. Neverthless, the comments of Tabashnik et al. (2004) are very helpful in elucidating the definitions of genotypic fitness used in Vacher et al. (2003) and in pointing out the essential factors in predicting the evolution of insect resistance to Bt transgenic crops, namely, accurate estimations of the fitness cost of resistance, of the dominance level of this cost, and of the variations in the dominance level of the advantage conferred by the resistance with Bt toxin dose.     

Can sugarcoated fingerprints be used to identify lurking viruses?

Comparative proteomics is increasingly used to detect biomarkers and therapeutic targets that differ between healthy and diseased populations; however, differences in posttranslational modifications have received less attention. In this issue, Yang et al. (Proteomics 2016, 16, 1872–1880) present data indicating that a glycoproteomics approach can detect N‐glycosylated membrane protein differences between non‐HIV‐infected and latently infected human CD4⁺ T‐cell lines, identifying 172 proteins differentially expressed by these cells. Latently infected CD4⁺ T cells are thought to represent the major barrier to eventual HIV cure, but do not express detectable levels of viral protein and have not been shown to express biomarkers that can distinguish them from the vastly more abundant uninfected CD4⁺ T‐cell population. The findings of Yang et al. suggest that glycoproteomic analyses may have untapped potential to identify novel biomarkers and therapeutic targets in cell populations not readily distinguishable be standard proteomic analyses.     

Cryopreservation of giant grouper Epinephelus lanceolatus (Bloch, 1790) sperm

The grouper, Epinephelus lanceolatus, is a vulnerable species of high economic value. An effective protocol was developed for the cryopreservation of E. lanceolatus by comparing different extenders produced by mixing various cryoprotectants (dimethyl sulfoxide, methanol and glycerol) and diluents (MPRS, TS‐2, TS‐19, Cortland and Hank's). Using computer‐assisted sperm analysis (CASA) and morphological analysis, the sperm motility and fertilization rates from post‐thaw sperm were comparable to untreated controls. The results revealed that MPRS (containing 12% DMSO) or TS‐19 (containing 12% DMSO), were the optimum extenders for protecting the sperm from cryo‐damage in liquid nitrogen. The post‐thaw sperm maintained high motility (90.61 ± 3.03%) and a fertilization rate (92.27 ± 2.43%) similar (P > 0.05) to fresh sperm (94.34 ± 4% and 94.10 ± 1.87%). This study is the first to report on the successful sperm cryopreservation of E. lanceolatus and provides an important tool for repopulating this species through aquaculture.