Targeting of a Germ Cell-specific Type 1 Hexokinase Lacking a Porin-binding Domain to the Mitochondria as Well as to the Head and Fibrous Sheath of Murine Spermatozoa

Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.

Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Descriptions of the mature spermatozoa of the lizards Crotaphytus bicinctores, Gambelia wislizenii (Crotaphytidae), and Anolis carolinensis (Polychrotidae) (Reptilia, Squamata, Iguania)

The spermatozoa of Crotaphytus bicinctores and Gambelia wislizenii (Crotaphytidae), and Anolis carolinensis (Polychrotidae) exhibit the squamate autapomorphies of a single perforatorium extending anteriorly from the apical tip of the paracrystalline subacrosomal cone, the presence of an epinuclear electron-lucent region, and extension of the fibrous sheath into the midpiece. Crotaphytid sperm differ from those of polychrotids in several respects, including: the structure of the perforatorium, the size of the epinuclear electron-lucent region, aspects of the acrosome complex, the arrangement and structure of intermitochondrial dense bodies, and in the distance the fibrous sheath extends into the midpiece. The sperm of C. bicinctores, G. wislizenii, and A. carolinensis are most similar to those of the agamids and phrynosomatids examined to date. No spermatozoal autapomorphies for Crotaphytidae or Polychrotidae were found. The condition of having the intermitochondrial dense bodies arranged in regular incomplete rings is tentatively defined as a synapomorphy of Iguania (although modified in Chamaeleonidae). Spermatozoal ultrastructure offers no characters that justify the separation of Iguanidae (sensu late) into several separate families. (C) 2001 Wiley-Liss, Inc.

Genetic male infertility and mutation of CATSPER ion channels.

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords 'CATSPER', 'male infertility', 'male contraception', 'immunocontraception' and 'pharmacologic contraception' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Anatomia de escapos de Tillandsia L. (Bromeliaceae) dos Campos Gerais do Paraná, Brasil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ultrastructure of the Spermatozoa of the Domestic Duck (Anas platyrhynchos sp.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electron microscopy study of lizard spermiogenesis in Tropidurus torquatus (Lacertilia)

In the course of an investigation on lizard spermiogenesis, the authors have observed events connected with the differentiation of the acrosome and middle-piece of the spermatozoa. Some differences from other reptilians were found as well as: the presence of a subacrosomal space, the absence of the fibrous sheath in the middle-piece, and a very short neck in the mature spermatozoa.

Eine neue Form der Pyruvatkinase in Säugerspermien

Nach Homogenisation ejakulierter Eberspermien und Zentrifugation des Homogenates blieben mehr als 60% der Aktivität des glykolytischen Enzyms Pyruvatkinase (PK) an Zellfragmenten im Sediment gebunden. Diese strukturgebundene PK wurde als PK-S bezeichnet. Das Detergenz Triton X-100 führte nicht zur Ablösung der PK-S; mit Trypsin konnten jedoch rund 80% der PK-S ohne Verlust an Aktivität von den Strukturen gelöst und durch kombinierte Kationenaustausch- und Hydrophobizitätschromatographie gereinigt werden (spezifische Aktivität: 116,7 U/mg Protein). Die lösliche PK aus Eberspermien konnte ebenfalls durch ein ähnliches Verfahren angereichert werden. Im Gel (SDS-PAGE) zeigten die Untereinheiten der PK-S mit 64.400 eine geringfügig größere relative Molekülmasse als die der PK-M1 aus Kaninchenmuskel (62.000). Die kinetischen Eigenschaften der abgelösten PK-S als auch der noch an Spermienstrukturen gebundenen PK-S und der löslichen PK aus Eberspermien waren sehr ähnlich und entsprachen der M1-Isoform der PK. Antikörper gegen Kaninchenmuskel-PK (Anti-PK-M1) reagierten auch mit der löslichen PK und der PK-S aus Eberspermien. Edman-Abbau der ersten 19 Aminosäuren zeigte, dass die tryptisch abgelöste PK-S am N-Terminus um 5 Aminosäuren gegenüber nativer PK-M1 verlängert ist, während der C-Terminus der erhaltenen PK-S-Sequenz mit einem meist nahe dem N-Terminus gelegenen Sequenzabschnitt der PK-M1 und -M2 übereinstimmt. Die N-terminale Verlängerung der nativen PK-S enthält sicherlich mehr als die nach tryptischer Lyse nachgewiesenen 5 Aminosäuren. Vergleiche der Aminosäure- und übersetzten Nukleotidsequenzen sowie die kinetischen Eigenschaften lassen vermuten, dass die PK-S, wie die PK-M1 und PK-M2, vom PKM-Gen codiert wird. Gegen die gereinigte PK-S wurden Antikörper in Kaninchen produziert. Da das Antiserum nicht ausreichend spezifisch für PK-S war, wurden aus ihm affinitätschromatographisch Antikörper (Anti-PK-S) isoliert, die hohe Affinität zu einem synthetisierten PK-S-Peptid (13 N-terminale Aminosäuren der tryptisch abgelösten PK-S) hatten. Dieses Anti-PK-S-Präparat war spezifisch für PK-S; es reagierte weder mit Kaninchenmuskel-PK noch mit löslicher PK oder anderen Proteinen aus Eberspermien. Anti-PK-S und Anti-PK-M1 wurden zur Lokalisierung von PK-S und löslicher PK in Spermien von Eber, Bulle und Mensch sowie in Schnitten von Eberhoden eingesetzt. Mit Anti-PK-S wurden der Bereich des Akrosoms und das lange flagellare Hauptstück sowie der Übergangsbereich zwischen Kopf und Mittelstück von Eberspermien fluoreszenzmarkiert, wogegen das kurze, die Mitochondrien enthaltende Mittelstück des Flagellums und der postakrosomale Kopfbereich nur mit Anti-PK-M1 markiert wurden. Immunogoldmarkierung in elektronenmikroskopischen Bildern bestätigte die Lokalisierung von PK-S im Akrosombereich. Im Hauptstück banden Anti-PK-M1 und Anti-PK-S an die fibröse Scheide. Glyzerinaldehyd-3-phosphat Dehydrogenase (GAPDH) konnte von mir ebenfalls im Akrosombereich, im Übergangsbereich zwischen Kopf und Mittelstück und an der fibrösen Scheide detektiert werden. Auch an Bullen- und Humanspermien konnte über Immunogoldmarkierung PK und vermutlich GAPDH an der fibrösen Scheide gezeigt werden. Im Akrosombereich dieser Spermien waren die Nachweise von PK und GAPDH jedoch nicht sicher. In Eberhodenschnitten war die PK-S erstmals, oder zumindest vermehrt, in den elongierenden Spermatiden über Fluoreszenzmarkierung nachweisbar, während andere, vermutlich somatische PK vermehrt in den früheren Stadien (Spermatogonien, aber auch in den Spermatozyten und runden Spermatiden) auftrat. Für die GAPDH zeigte sich ein ähnlicher Entwicklungsverlauf. Die Ergebnisse zeigen, dass in Eberspermien zwei Isoformen der PK auftreten: eine N-terminal verlängerte, strukturgebundene Form, die PK-S, und eine lösliche Form, die beide der PK-M1 ähneln. Der ungewöhnliche N-Terminus der PK-S dient vermutlich der spezifischen räumlichen Anordnung der PK-S im Akrosombereich und an der fibrösen Scheide, nicht aber der Modulation kinetischer Eigenschaften. Meine Untersuchungen stützen die Hypothese, dass in bestimmten Kompartimenten von Säugerspermien die Glykolyse durch Verankerung einiger ihrer Enzyme strukturell hochgeordnet ist. Dadurch wird vermutlich die Versorgung der Mitochondrien-freien Regionen mit ATP sichergestellt. Man kann diese Organisation als Anpassung des Stoffwechsels von Spermien deuten, bei denen die Mitochondrien in einem kleinen Bereich (Mittelstück) hinter dem Spermienkopf kompartimentiert sind. Im Hauptstück des Flagellums könnte die Glykolyse ATP für die Spermienmotilität liefern, im Akrosombereich für die Verhinderung einer vorzeitigen Akrosomreaktion. Somit käme der strukturierten Glykolyse eine essentielle Bedeutung für die Befruchtungsfähigkeit von Säugerspermien zu.

Ultrastructure of the spermatozoon of Apus apus (Linnaeus 1758), the common swift (Aves; Apodiformes; Apodidae), with phylogenetic implications

The spermatozoon of Apus apus is typical of non-passerines in many respects. Features shared with palaeognaths and the Galloanserae are the conical acrosome, shorter than the nucleus; the presence of a proximal as well as distal centriole; the elongate midpiece with mitochondria grouped around an elongate distal centriole; and the presence of a fibrous or amorphous sheath around the principal piece of the axoneme. The perforatorium and endonuclear canal are lost in A. apus as in some other non-passerines. All non-passerines differ from palaeognaths in that the latter have a transversely ribbed fibrous sheath whereas in non-passerines it is amorphous, as in Apus, or absent. The absence of an annulus is an apomorphic but homoplastic feature of swift, psittaciform, gruiform and passerine spermatozoa. The long distal centriole, penetrating the entire midpiece, is a remarkably plesiomorphic feature of the swift spermatozoa, known elsewhere only in palaeognaths. The long centriole of Apus, if not a reversal, would be inconsistent with the former placement of the Apodiformes above the Psittaciformes from DNA-DNA hybridization. In contrast to passerines, in A. apus the microtubules in the spermatid are restricted to a transient single row encircling the cell. The form of the spermatozoon fully justifies the exclusion of swifts from the passerine family Hirundinidae.

Ultrastructure of the spermatid of Caprimuigus europaeus Linnaeus 1758, the European Nightjar (Aves; Caprimulgidae), with phylogenetic implications

The sperm of Caprimulgus europaeus is typical of other nonpasserines in many respects. Features shared with Paleognathae and Galloanserae are the conical acrosome, shorter than the nucleus; the presence of a perforatorium and endonuclear canal; the presence of a proximal as well as distal centriole; the elongate midpiece with mitochondria grouped around a central axis (here maximally six mitochondria in similar to 10 tiers); and the presence of a fibrous or amorphous sheath around the principal piece of the axoneme. A major (apomorphic) difference from paleognaths and galloanserans is the short distal centriole, the midpiece being penetrated for most of its length by the axoneme and for only a very short proximal portion by the centriole. Nonpasserines differ from paleognaths in that the latter have a transversely ribbed fibrous sheath, whereas in nonpasserines it is amorphous, as in Caprimulgus, or absent. The absence of an annulus is an apomorphic feature of Caprimulgus, apodiform, psittaciform, gruiform, and passerine sperm, homoplastic in at least some of these. In contrast to passerines, in Caprimulgus the cytoplasmic microtubules in the spermatid are restricted to a transient longitudinal manchette. The structure of the spermatid and spermatozoon is consistent with placement of the Caprimulgidae near the Psittacidae, but is less supportive of close proximity to the Apodidae, from DNA-DNA hybridization and some other analyses.

Meningial perineurioma: a benign peripheral nerve sheath tumor in a previously unrecognized central nervous system location, mimicking meningioma

Perineurioma is an uncommon, mostly benign, spindle-cell tumor of peripheral nerve sheath origin with a predilection for the soft tissues. Although increasing awareness points to the sites of involvement by perineurioma possibly being as ubiquitous as those frequented by schwannian tumors, only one intracerebral example has been described to date. We report on a surgically resected perineurioma of the falx cerebri in an 86-year-old woman. Preoperative imaging showed an enhancing extraaxial mass of 6 cm × 5.7 cm × 3.7 cm. Histologically, the tumor consisted of a proliferation of spindle cells interwoven by a lattice of basal lamina. Alongside a prevailing soft tissue perineurioma pattern, sclerosing and reticular areas were seen as well. Tumor cells coexpressed EMA and GLUT-1, and a minority immunoreacted for smooth muscle actin. Pericellular basal lamina was decorated with collagen type IV. No staining for S100 protein was detected. Mitotic activity was virtually absent, and the MIB1 labeling index averaged 2%. Ultrastructural examination revealed abundant pinocytotic vesicles within and conspicuous tight junctions between slender cytoplasmic processes which, in turn, were encased by discontinuous basal lamina. FISH analysis confirmed loss of at least part of one chromosome 22q. This observation calls attention to perineurioma as a novel item in the repertoire of low-grade meningial spindle cell neoplasms, in the differential diagnostic context of which it is apt to being misconstrued as either meningioma, solitary fibrous tumor, or neurofibroma. Confusion with the latter bears the risk of overgrading innocuous features of perineurioma as criteria for malignancy.

Changes In The Connective Tissue Sheath Of Wistar Rat Nerve With Aging.

The alterations due to aging in the peripheral nerves can affect the physiology of these structures. Thus, the purpose of the present study was to describe the activity of the MMP-2 and MMP-9, as well as the structure and composition of the extracellular matrix of the rat sciatic nerve during maturation and aging. Our results have shown that the extracellular matrix of the sciatic nerve of 30-, 180- and 730-day-old Wistar rats present ultrastructural, morphometrical and biochemical changes during aging. The perineurium was the structure most affected by age, as evidenced by a decrease in thickness and in collagen fibril content. Cytochemical analysis detected proteoglycans in the basal membrane of Schwann cells and around perineural cells, as well as on the collagen fibrils of the perineurium and endoneurium at all ages. Biochemical analyses showed that the quantity of non-collagenous proteins was higher in 730-day-old animals compared to other ages, while the uronic acid content was higher in 30-day-old animals. Morphometrical analysis detected greater numbers of myelinated fibers and increased myelin thickness in 180-day-old animals. Zymography analysis detected greater amounts and activity of MMP-2 and MMP-9 in 180- and 730-day-old animals compared to younger rats. In conclusion, our results showed changes in the structural organization and composition of extracellular matrix of the sciatic nerve during aging, such as increase in the non-collagenous protein content and higher MMP-2 and MMP-9 activity, decrease in uronic acid concentration and in collagen fibril content in the perineurium, as well as degeneration of nerve fibers.

Macropore sheath: quantification of plant root and soil macropore association

Plants require roots to supply water, nutrients and oxygen for growth. The spatial distribution of roots in relation to the macropore structure of the soil in which they are growing influences how effective they are at accessing these resources. A method for quantifying root-macropore associations from horizontal soil sections is illustrated using two black vertisols from the Darling Downs, Queensland, Australia. Two-dimensional digital images were obtained of the macropore structure and root distribution for an area 55 x 55 mm at a resolution of 64 mu m. The spatial distribution of roots was quantified over a range of distances using the K-function. In all specimens, roots were shown to be clustered at short distances (1-10 mm) becoming more random at longer distances. Root location in relation to macropores was estimated using the function describing the distance of each root to the nearest macropore. From this function, a summary variable, termed the macropore sheath, was defined. The macropore sheath is the distance from macropores within which 80% of roots are located. Measured root locations were compared to random simulations of root distribution to establish if there was a preferential association between roots and macropores. More roots were found in and around macropores than expected at random.

Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug

A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia, The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794(T) (= DSM 12717(T)).

Reticular angiomatoid ""malignant"" fibrous histiocytoma-a case report with cytogenetics and molecular genetic analyses

Angiomatoid ""malignant"" fibrous histiocytoma is a rare sarcoma of low malignant potential that occurs most commonly in the extremities of children and young adults. Herein, we present a case of angiomatoid malignant fibrous histiocytoma with unusual histologic features arising in the mediastinum of an 80-year-old man. The tumor exhibited a reticular growth pattern and myxoid stroma. The tumor cells expressed epithelial membrane antigen and desmin. Cytogenetic analysis revealed the translocation t(2;22)(q33;q12). Molecular genetic analysis confirmed the rearrangement of the EWSR1 locus and the presence of the EWSR1/CREB1 fusion. This report expands the clinicopathologic spectrum of angiomatoid malignant fibrous histiocytoma and underscores the value of integrating morphologic, immunophenotypic, and molecular findings in the identification of its unusual morphologic variants. (C) 2011 Elsevier Inc. All rights reserved.