Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.     

Altérations morphologiques des spermatozoïdes en microscopie électronique: indications, phénotypes, fécondance, et pronostic de fertilité

Conventional semen analysis (sperm count) is limited to examination of spermatozoa at a magnification of x1,000, which may be insufficient in rare situations. Electron microscopy sperm examination allows high-power (x 100,000) analysis of sperm organelles and quantification of abnormalities of the constituents involved in sperm mobility and fertility potential. Electron microscopy sperm morphology examination is rarely indicated and is reserved to: 1) severe monomorphic and stable teratospermia (globozoospermia = spermatozoa with a round head and no acrosome, pinheads = decapitated spermatozoa), 2) partial (asthenospermia) or total (akinetospermia) alteration of sperm mobility and/or quality of sperm movement. All of these anomalies are associated with primary infertility. Globozoospermia and pinheads can be detected by light microscopy. Electron microscopy sperm morphology examination precisely identifies and quantifies sperm abnormalities. Pathological phenotypes have a heterogeneous expression. The organelles of spermatozoa other than those primarily involved in the pathological phenotype may also present alterations. Globozoospermia is generally characterized by the absence of elongation of the nucleus, and absence of the acrosome and the post-acrosomal region. The implantation fossa and basal plate are generally missing in decapitated spermatozoa. Asthenospermia may be an indication for electron microscopy sperm examination when it is not associated with necrospermia. Sperm with fibrous sheath dysplasia (FSD) generally present a short flagella and very low overall mobility, less than 5%. The various phenotypes are characterized by abnormal arrangements of the constituents of the fibrous sheath and 20% of patients also present respiratory tract disease. In primary ciliary dyskinesia (PCD), spermatozoa are often immobile and present a normal morphology on light microscopy. Apart from the complete form with absent axoneme, incomplete forms are also observed with absence of the dynein arms, peripheral doublets, microtubules. These phenotypes have a low prevalence in the population of infertile men. A familial incidence, parental consanguinity, and a high incidence in certain geographical regions are frequently reported, suggesting the existence of one or several genetic mechanisms. Despite the limited state of knowledge at the present time, couples must be informed about the possible transmission of the phenotype to their descendants. All men with these phenotypes are spontaneously infertile. The only alternative fertilization technique is intracytoplasmic sperm injection (ICSI). According to the literature and our own experience, the results of ICSI with sperm presenting these phenotypes are poorer than those of ICSI in general. Electron microscopy is not only a diagnostic tool in severe male infertility, but also a prognostic indicator of the success of management by ICSI, which must be evaluated for each case.     

Optimal design of thermally stable proteins

MOTIVATION: For many biotechnological purposes, it is desirable to redesign proteins to be more structurally and functionally stable at higher temperatures. For example, chemical reactions are intrinsically faster at higher temperatures, so using enzymes that are stable at higher temperatures would lead to more efficient industrial processes. We describe an innovative and computationally efficient method called Improved Configurational Entropy (ICE), which can be used to redesign a protein to be more thermally stable (i.e. stable at high temperatures). This can be accomplished by systematically modifying the amino acid sequence via local structural entropy (LSE) minimization. The minimization problem is modeled as a shortest path problem in an acyclic graph with nonnegative weights and is solved efficiently using Dijkstra's method.     

The Nature of Urban Soils and Their Role in Ecological Restoration in Cities

Current and predicted trends indicate that an increasing proportion of the world’s population is living in urban and suburban places. The nature of the urban environment becomes an important factor if we are concerned with the restoration and preservation of biodiversity and ecosystems in and around cities. This article highlights the varied impacts of cities on soils and their implications for restoration planning and expectations of restoration “success.” Urban soils exist in different historical and formational trajectories than their local nonurbanized counterparts due to direct anthropogenic disturbance and indirect environmental impacts from urbanization. Therefore, urban soils often exhibit altered physical, chemical, and biological characteristics in comparison to local nonurbanized soils. Several unique features of urban soils and urban ecosystems pose particular issues for ecological restoration or the improvement of degraded soil conditions in cities. The creation of novel soil types, conditions that promote invasion by non‐natives, the strong influence of past land use on soil properties, and the presence of strong interactions and alternative stable states set up unique difficulties for the restoration of urban soils. Soils in urban restorations are a medium that can be deliberately manipulated to improve site conditions or in the monitoring of soil conditions as indices of ecosystem status. Including an explicit role for strong manipulations of soils in urban ecosystems changes how we approach baselines, management, and reference conditions in urban ecological restoration. With an understanding of urban soil ecological knowledge, we can guide aspects of urban ecological restoration toward successful outcomes.     

Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation.

Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.     

Time to chill: effects of simulated global change on leaf ice nucleation temperatures of subarctic vegetation

We investigated the effects of long-term (7-yr) in situ CO(2) enrichment (600 μmol/mol) and increased exposure to UV-B radiation, the latter an important component of global change at high latitudes, on the ice nucleation temperatures of leaves of several evergreen and deciduous woody ericaceous shrubs in the subarctic (68° N). Three (Vaccinium uliginosum, V. vitis-idaea, and Empetrum hermaphroditum) of the four species of shrubs studied showed significantly higher ice nucleation temperatures throughout the 1999 growing season in response to CO(2) enrichment and increased exposure to UV-B radiation relative to the controls. The same species also showed a strong interactive effect when both treatments were applied together. In all cases, leaves cooled to below their ice nucleation temperatures failed to survive the damage resulting from intracellular ice formation. Our results strongly suggest that future global change on a decadal time scale (atmospheric CO(2) increases and polar stratospheric O(3) destruction) will lead to increased foliage damage of subarctic vegetation by severe late spring or early autumnal frosting events. Indeed, in support of our experimental findings, there is now some evidence that increases in atmospheric CO(2) concentration over the past three to four decades may already have acted in this manner on high-elevation arboreal plants in the Swedish Scandes. The implications for vegetation modeling in a future "greenhouse" world and palaeoclimate estimates from high-latitude plant fossils dating to the high-CO(2) environment of the Mesozoic are discussed.     

Root to shoot ratio of crops as influenced by CO2

Crops of tomorrow are likely to grow under higher levels of atmospheric CO₂. Fundamental crop growth processes will be affected and chief among these is carbon allocation. The root to shoot ratio (R:S, defined as dry weight of root biomass divided by dry weight of shoot biomass) depends upon the partitioning of photosynthate which may be influenced by environmental stimuli. Exposure of plant canopies to high CO₂ concentration often stimulates the growth of both shoot and root, but the question remains whether elevated atmospheric CO₂ concentration will affect roots and shoots of crop plants proportionally. Since elevated CO₂ can induce changes in plant structure and function, there may be differences in allocation between root and shoot, at least under some conditions. The effect of elevated atmospheric CO₂ on carbon allocation has yet to be fully elucidated, especially in the context of changing resource availability. Herein we review root to shoot allocation as affected by increased concentrations of atmospheric CO₂ and provide recommendations for further research. Review of the available literature shows substantial variation in R:S response for crop plants. In many cases (59.5%) R:S increased, in a very few (3.0%) remained unchanged, and in others (37.5%) decreased. The explanation for these differences probably resides in crop type, resource supply, and other experimental factors. Efforts to understand allocation under CO₂ enrichment will add substantially to the global change response data base.Abbreviations R:S root to shoot ratio, dry weight basis     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.