bluuebunny/crossref_metadata_2025 huggingface dataset:
Poroelastic properties of bovine vertebral trabecular bone
Authors: Lim Tae‐Hong , Hong Jung Hwa
Published: February 2005

The theory of poroelasticity has been used to study bone mechanics without directly measuring poroelastic properties. In this study, we developed an experimental protocol and measured the poroelastic properties of bovine vertebral trabecular bone. Mean (±SD) values for drained shear modulus, drained Poisson's ratio, undrained Poisson's ratio, Skempton's coefficient, and permeability coefficient were, respectively, 90.85 (±59.59) MPa and 0.242 (±0.099), 0.399 (±0.083), 0.0851 (±0.144), and 16.31 (±8.02) × 10−8m2/Pa/sec, respectively. The experimental protocol can be used generally for the measurement of poroelastic properties of bone when cylindrical specimens are available. Measured poroelastic properties can be used directly or converted to Biot's coefficient and modulus, without assuming the incompressibility of solid and fluid constituents, for the poroelastic modeling of bone.
Mechanobiology and joint conformity regulate endochondral ossification of sesamoids
Authors: Sarin Vineet K. , Carter Dennis R.
Published: February 2005

Sesamoid bones form by the endochondral ossification of sesamoid cartilages. This ossification process is thought to be similar to that responsible for the formation of secondary ossific nuclei in long‐bone epiphyses. Sesamoids ossify much later in development than do epiphyses, however, and bone formation within sesamoids often begins by way of multiple ossific nuclei. Endochondral growth and ossification in the formation of secondary ossific nuclei have previously been correlated with distributions of the octahedral shear and hydrostatic stresses generated in vivo within cartilage anlagen. In this study, we used two‐dimensional finite element analysis to predict the distributions of octahedral shear and hydrostatic stresses in an idealized model of a sesamoid cartilage subjected to in vivo loading. We examined the influence of sesamoid joint conformity. The distribution of an osteogenic stimulus was calculated with an approach similar to that used to predict epiphyseal ossification. The results suggest that, compared with conforming joints, nonconformity between the sesamoid cartilage and its articulating surface, which arises during early development, produces higher contact pressures within the sesamoid and leads to a thicker articular cartilage layer. For a nonconforming joint surface, the results suggest that ossification is favored anywhere within a broad internal region of the sesamoid, whereas a layer at the articular surface will remain cartilaginous. These findings highlight the subtle differences between ossification processes in epiphyses and sesamoids, indicating that the mechanical stress environment in sesamoids produces a diffuse stimulus leading to the onset of ossification and that the degree of joint nonconformity may influence the thickness of the articular cartilage layer.
Polysulphated glycosaminoglycan treatments can mitigate decreases in stiffness of articular cartilage in a traumatized animal joint
Authors: Ewers B. J. , Haut R. C.
Published: February 2005

A single, blunt impact to the rabbit patellofemoral joint has been shown to decrease the stiffness of retropatellar cartilage and increase the thickness of the underlying bone. Polysulphated glycosaminoglycan treatments, on the other hand, have been shown to inhibit the degradation of articular cartilage and possibly increase synthesis of collagen and glycosaminoglycans in experimental studies on diseased joints. The aim of the current study was to examine the effect of early treatments with polysulphated glycosaminoglycans on cartilage using anin vivopost‐trauma animal model. The study used 24 Flemish Giant rabbits in three groups: control, impacted, and impacted with treatment. Treatment consisted of intramuscular injections the day of insult and every 4 days thereafter for 6 weeks. At 30 weeks after trauma, mechanical tests were performed on the retropatellar cartilage to determine its mechanical stiffness. The patellae were also grossly evaluated for surface lesions on the retropatellar cartilage and histologically processed to measure the thickness of the subchondral bone. The rabbits that received no treatment had a statistically significant decrease in stiffness (modulus) for the cartilage of the impacted patellae compared with that of the contralateral, unimpacted patellae and compared with the cartilage of rabbits in the control group. The degradation in mechanical stiffness, however, was not observed in patellae of rabbits in the group receiving treatment. There was also a significant increase in the underlying thickness of the subchondral plate on the impacted patellae compared with that on the contralateral, unimpacted sides for rabbits in both the treated and nontreated groups. In conclusion, the polysulphated glycosaminoglycan treatments minimized a decrease in mechanical stiffness (modulus) of retropatellar articular cartilage 30 weeks after trauma. The mechanism by which the mechanical stiffness of the cartilage was preserved is unknown.
Mixture of hyaluronic acid and phospholipid prevents adhesion formation on the injured flexor tendon in rabbits
Authors: Moro‐Oka Takaaki , Miura Hiromasa , Mawatari Taro , Kawano Tsutomu , Nakanishi Yoshitaka , Higaki Hidehiko , Iwamoto Yukihide
Published: February 2005

Dipalmitoyl phosphatidylcholine, a highly surface‐active polar lipid, has been implicated as a potential boundary lubricant for synovial joints. We examined the effects of dipalmitoyl phosphatidylcholine on the flexor tendon and its protective effect against postoperative adhesion in two experimental steps. First, the flexor digitorum fibularis and the distal pulley of rabbits were set for a friction test. The test was performed with saline solution, sodium hyaluronate, or a mixture of dipalmitoyl phosphatidylcholine and sodium hyaluronate as the lubricant. The friction coefficient was significantly lower with the mixture of dipalmitoyl phosphatidylcholine and sodium hyaluronate than with saline solution or sodium hyaluronate. We concluded that the decreased friction coefficient indicates that dipalmitoyl phosphatidylcholine could complement the boundary‐lubricating ability of the tendon. In the second experiment, we used an experimental adhesion model of the flexor digitorum fibularis in the rabbit. During the operation, either saline solution, sodium hyaluronate, or a mixture of dipalmitoyl phosphatidylcholine and sodium hyaluronate was injected into the tendon sheath. The specimen was sent to another tester, and the work required to tear off the adhesion was measured. The work required was significantly greater for the tendons that had been injected with saline solution than for those given injections of dipalmitoyl phosphatidylcholine and sodium hyaluronate. Our findings suggest that dipalmitoyl phosphatidylcholine plays an important role in the boundary lubrication of the tendon and that after tendon injury, the administration of a mixture of dipalmitoyl phosphatidylcholine and sodium hyaluronate may improve tendon lubrication and prevent adhesion formation.
Quantitative small‐animal surrogate to evaluate drug efficacy in preventing wear debris‐induced osteolysis
Authors: Schwarz Edward M. , Benz Eric B. , Lu Allen P. , Goater J. Jeffrey , Mollano Anthony V. , Rosier Randy N. , Puzas J. Edward , O'Keefe Regis J.
Published: February 2005

Individuals who suffer from severe joint destruction caused by the various arthritidies often undergo total joint arthroplasty. A major limitation of this treatment is the development of aseptic loosening of the prosthesis in as many as 20% of patients. The current paradigm to explain aseptic loosening proposes that wear debris generated from the prosthesis initiates a macrophage‐mediated inflammatory response by resident macrophages, leading to osteoclast activation and bone resorption at the implant interface. No therapeutic interventions have been proved to prevent or inhibit aseptic loosening. The development of therapeutic strategies is limited due to the absence of a quantitative surrogate in which drugs can be screened rapidly in large numbers of animals. We have previously described a model in which titanium particles implanted on mouse calvaria induce an inflammatory response with osteolysis similar to that observed in clinical aseptic loosening. Here, we present new methods by which the osteolysis in this model can be quantified. We determined that 6–8‐week‐old mice in normal health have a sagittal suture area of 50 (±6) μm2, which contains approximately five osteoclasts. As a result of the titanium‐induced inflammation and osteolysis, the sagittal suture area increases to 197 (±27) μm2, with approximately 30 osteoclasts, after 10 days of treatment. The sagittal suture area and the number of osteoclasts in the calvaria of sham‐treated mice remained unchanged during the 10 days. We also determined the effects of pentoxifylline, a drug that blocks the responses of tumor necrosis factor‐α to wear debris, and the osteoclast inhibitor alendronate. We found that both drugs effectively block wear debris‐induced osteolysis but not osteoclastogenesis. In conclusion, we found the measurements made with this model to be reproducible and to permit quantitative analysis of agents that are to be screened for their potential to prevent aseptic loosening.
Biomechanical regulation of matrix metalloproteinase‐9 in cultured chondrocytes
Authors: Jin Gang , Sah Robert L. , Li Yi‐Shuan , Lotz Martin , Shyy John Y‐J. , Chien Shu
Published: February 2005

Abnormal mechanical loading of joints may induce degeneration of articular cartilage. Shear stress is one mode of mechanical loading that may regulate chondrocyte metabolism. We investigated the mechanism by which shear stress induces the gene encoding matrix metalloproteinase‐9, a mediator of the progressive degradation of articular cartilage in osteoarthritis. In vitro experiments using passaged rabbit chondrocytes in monolayer culture subjected to a shear stress of 16 dyn/cm2 (1.6 Pa) in a flow channel showed increased expression of the matrix metalloproteinase‐9 gene. The induction of matrix metalloproteinase‐9 appeared to depend on a region in the 5′ promoter of the gene that contains a 12–0‐tetradecanoylphorbol 13‐acetateresponsive element. Transfection experiments using a construct containing a luciferase reporter driven by a 12–0‐tetradecanoylphorbol 13‐acetate‐responsive element indicated that shear stress activated a 12–0‐tetradecanoylphorbol 13‐acetate‐responsive element‐mediated transcription in chondrocytes. Similar experiments showed that shear stress induced a matrix metalloproteinase‐9 promoter construct (matrix metalloproteinase‐9‐luciferase). Shear stress activated c‐Jun NH2‐terminal kinase, extracellular signal‐regulated kinase, and p38. Transfection o, matrix metalloproteinase‐9‐luciferase together with the dominant negative mutant of c‐Jun NH2‐terminal kinase, but not with that of extracellular signal‐regulated kinase or p38, attenuated the shearinduced matridx metalloproteinase‐9 promoter activity. In addition, transfection of constructs encoding dominant negative mutants of Ras, Rac, and Cdc42 attenuated the induction of c‐Jun transcriptional activity by shear stres. Thus, shear stimulation of chondrocytes stimulates Ras, Rac, and Cdc42, which subsequently activate c‐Jun NH2‐terminal kinase to induce a 12–0‐tetradecanoylphorbol 13‐acetate‐responsive elementmediated expression of matrix metalloproteinase‐9.
Delamination rates of tissue flaps used in articular cartilage repair
Authors: Driesang Iris M. K. , Hunziker Ernst B.
Published: February 2005

A popular strategy in the treatment of articular cartilage lesions involves the introduction of cell suspensions into the defect void and its closure with a periosteal flap (autologous chondrocyte transplantation technique). We applied this methodology in goats and discovered that all sutured flaps (n = 6 animals) became detached from nonimmobilized joints during the recovery period. The purpose of this study was to ascertain whether postoperative restriction of joint movement could prevent the delamination of flaps. Partial‐thickness defects were created in the knee joint cartilage of 27 goats. These defects were then filled with a fibrin matrix and covered with periosteal (n = 6) or fascial (n = 21) flaps, which were sutured with simple, interrupted stitches to the surrounding tissue. The joints were immobilized by means of a modified Robert Jones bandage for periods of 2–6 weeks, after which time they were inspected for the absence or presence of flaps. In four animals, joint immobilization for 3 weeks was followed by free movement for a similar period. Four of the six periosteal flaps and two of the 21 fascial ones became delaminated after the period of immobilization. In the four goats permitted 3 weeks of free joint movement following a similar period of joint immobilization, all flaps (which had been retained up to the end of the immobilization period) became detached. These findings indicate that joint immobilization hinders the delamination of flaps but that this restriction of movement must be sustained for an undefined period of time. The nature of the tissue used for flaps also influences their rate of retention by immobilized joints.
Quantitative assessment of in vivo bone regeneration consolidation in distraction osteogenesis
Authors: Windhagen Henning , Kolbeck Stefan , Bail Hermann , Schmeling Arno , Raschke Michael
Published: February 2005

We present a new method for quantitatively assessing the consolidation of bone regeneration by performing distraction osteogenesis in micropigs. We measured in vivo stiffness using a newly developed, revolving, bone‐healing meter. After the micropigs were killed, we obtained maximum torsional moment data for the regenerated bones by destructive mechanical testing, and we then correlated these data with the data for stiffness. We found a highly significant regression between in vivo stiffness and maximum torsional moment (r2 = 0.80), suggesting that monitoring stiffness may be useful for the prediction of bone regeneration in distraction osteogenesis. Therefore, our method may be a reliable tool for future quantitative monitoring of healing progress in patients with healing bones or in animal studies addressing treatments to increase bone formation in long‐bone defects.
Osteoprogenitor cells within skeletal muscle
Authors: Bosch Patrick , Musgrave Douglas S. , Lee Joon Yung , Cummins Jim , Shuler Franklin , Ghivizzani Steven C. , Evans Chris , Robbins Paul D. , Huard Johnny
Published: February 2005

The formation of ectopic bone within skeletal muscle is a widely observed phenomenon. However, the source of the osteoprogenitor cells responsible for ectopic bone formation remains unknown. This study was designed to test for osteogenic differentiation among cells isolated from skeletal muscle tissue. Different subpopulations of cells derived from an adult mouse skeletal muscle were tested for induction of alkaline phosphatase activity after exposure to bone morphogenetic protein‐2 in vitro. A responsive subpopulation was identified, transduced with a retrovirus encoding for β‐galactosidase (Rv‐lacZ) and an adenoviral construct encoding for one bone morphogenetic protein‐2, and injected into the hindlimb of immune compromised (severe combined immunodeficient, or SCID) mice. The injected cells appeared to actively participate in the ectopic bone formation. The existence of lacZ‐positive muscle‐derived cells colocalized with osteocalcin‐producing cells within lacunae of newly formed bone matrix suggests osteoblast and osteocyte differentiation. Although a specific cell was not isolated, these data support the contentions that osteoprogenitor cells reside within skeletal muscle and that muscle may represent a source other than bone marrow for the harvest of these cells.
The influence of frying medium degradation on fat uptake and texture of French fries
Authors: Kita Agnieszka , Lisińska Grażyna , Powolny Małgorzata
Published: February 2005

The purpose of the present investigation was to study the influence of the degree of degradation of different kinds of oils used as a frying medium on fat uptake and texture of frozen pre‐fried French fries. As has been found, the degradation degree of the frying medium depends on the kind of oil and processing time. Liquid hydrogenated rapeseed oil exhibited the best thermo‐oxidative stability among the oils under investigation. The kind of oil influenced fat uptake and the texture of French fries. The lowest fat uptake was observed with French fries fried in solid oil. Some correlations between fat uptake and changes in fatty acid content were found in the frying medium during frying. Fat absorption increased with increasing unsaturated fatty acids and decreasing saturated fatty acid content. The texture of French fries fried in hydrogenated oils was harder than that exhibited by French fries fried in liquid rapeseed oil. The hardness of French fries fried in liquid oils decreased during frying while the hardness of French fries fried in solid oil increased. Some correlations between the texture of French fries and iodine value and fatty acids content of frying media were found. The hardness of French fries increased with increasing content of saturated fatty acids and decreasing unsaturated fatty acids and trans isomer fatty acid content. Copyright © 2005 Society of Chemical Industry
Rheological and Structural Investigation of Layered Silicate Nanocomposites Based on Polyamide or Polyethylene: Influence of Processing Conditions and Volume Fraction Effects
Authors: Médéric Pascal , Razafinimaro Tolotrahasina , Aubry Thierry , Moan Michel , Klopffer Marie‐Hélène
Published: February 2005

Organoclay polymer nanocomposites offer improved material properties at very low filler loadings making them of immediate interest for industrial applications. In this study two matrices are used : a polar polymer, PA12, and a non polar polymer, LLDPE. Many articles focused on the importance of the chemistry used to modify the surface of the clay. Numerous papers studied the influence of the compatibilizing agent necessary to obtain intercalated structure, when the matrix is non polar. This paper demonstrates the importance of the way the clay was melt processed into the thermoplastic matrix. Layered silicate nanocomposites based on PA12 or LLDPE were characterized by X‐ray diffraction, transmission electron microscopy and rheological tests. The influence of volume fraction on rheological and structural properties is also investigated.
Starch Nanocrystal Fillers in an Acrylic Polymer Matrix
Authors: Angellier Hélène , Putaux Jean‐Luc , Molina‐Boisseau Sonia , Dupeyre Danielle , Dufresne Alain
Published: February 2005

Aqueous suspensions of nanocrystals can be obtained by acid hydrolysis of native waxy maize starch granules. The disruption of waxy maize starch granules by 2.2N HCl hydrolysis has been followed by scanning and transmission electron microscopy and laser granulometry. The mechanical properties of composite materials made of poly(styrene‐co‐butyl acrylate) filled with starch nanocrystals were characterized by dynamic mechanical analysis. These nanocrystals appeared to be an interesting reinforcing phase in a thermoplastic matrix.
Polymer Layered Silicate/Carbon Nanotube Nanocomposites: Morphological and Rheological Properties
Authors: Peeterbroeck S. , Alexandre M. , Nagy J.B. , Moreau N. , Destrée A. , Monteverde F. , Rulmont A. , Jérôme R. , Dubois Ph.
Published: February 2005

Morphological and rheological properties of new ternary nanocomposites based on ethylene vinyl acetate copolymers (EVA), commercial organo‐modified clays (organoclays) and purified multi‐walled carbon nanotubes (MWNTs), prepared via direct melt blending, have been evaluated. For sake of comparison, the corresponding binary compositions, i.e., EVA filled with either organoclays or MWNTs, have been investigated as well. While extensive exfoliation can be observed for binary EVA/clay nanocomposites, the addition of MWNTs appears to limit clay exfoliation. Rheological properties show that both clay and MWNTs increase the elastic modulus of the nanocomposites, reflecting the high degree of nanoparticle interconnectivity that can be found in these materials.
Synthetic Hydromagnesite as Flame Retardant. A Study of the Stearic Coating Process
Authors: Haurie Laia , Fernández Ana Inés , Velasco José Ignacio , Chimenos Josep Maria , Ticó‐Grau Josep Ramón , Espiell Ferran
Published: February 2005

A synthetic hydromagnesite obtained from an industrial by‐product rich in magnesium oxide was employed and evaluated as a non‐halogenated flame retardant for poly(ethylene‐co‐vinyl acetate).

The filler was characterized with different techniques (such as specific surface area, TGA, particle morphology and size measurements, WAXS). Significant differences were found between the synthetic hydromagnesite and the natural one.

Synthetic hydromagnesite was coated with stearic acid and the effectiveness of the coating process was studied by the dye adsorption method and sedimentation volume measurements. The amount of coating agent ranged from 1 to 4.5%. This factor was found to have a significant effect on the thermal decomposition behaviour of the filler.

A poly(ethylene‐co‐vinyl acetate) (27% of VA) was filled with the coated synthetic grades of hydromagnesite as well as with two commercial flame retardants and different physicochemical properties were evaluated, including their flame retardant effect.
Treatment of SMC Composite Waste for Recycling as Reinforcing Fillers in Thermoplastics
Authors: Perrin Didier , Leroy Eric , Clerc Laurent , Bergeret Anne , Lopez‐Cuesta José‐Marie
Published: February 2005

Needs for recycling thermoset composite materials are considerably increasing. Unfortunately, such materials are particularly difficult to recycle due to their crosslinked nature. In this paper, we focus on the case of Sheet Moulding Compounds (SMC), which consist of an unsaturated polyester filled with glass fibres and calcium carbonate. We consider the possibilities of mechanical treatments allowing to obtain ground fractions that could be used as fillers for the reinforcement of thermoplastics. Two processes are described and compared with an existing industrial one. Finally, optimised fractions of SMC waste are introduced into polypropylene, leading to promising results in term of mechanical properties.
Modified Cellulose Fibers as Reinforcing Fillers for Macromolecular Matrices
Authors: Gandini Alessandro , Belgacem Mohamed Naceur
Published: February 2005

Various cellulose substrates were submitted to different surface OH chemical modifications in non‐swelling media and then fully characterised by FTIR, XPS, contact angle measurements and elemental analysis. The alternative procedures adopted were:

(i) simple grafting with monomeric or oligomeric agents which bore one or several reactive functions, either as a single terminal moiety or as numerous moieties placed along the chain;

(ii) grafting with polymerisable molecules, which involved the use of reagents of small molecular size, but bearing two functions, one able of reacting with the surface cellulose OH groups and the other used as a source of subsequent covalent linkage with the matrix;

(iii) the use of planar stiff molecular configurations bearing two identical reactive functions, calling upon the working hypothesis that only one function will react with a cellulose OH group, whereas the other will be left to copolymerise with the matrix;

(iv) coupling with siloxanes using the same strategy as in (ii), except for the fact that this specific approach involves molecules already commonly used in glass fibres treatment;

(v) grafting with organometallics such as triethyl aluminium following the same working hypothesis as in (iii) but with a second reaction involving monomeric or polymeric alcohols or amines.
Reply: Bilateral chorea–ballism associated with hyperthyroidism
Authors: Kostic Vladimir S.
Published: February 2005

The original letter to which this reply refers was published in Movement Disorders Mov Disord (2005) 20 (4) 512.
More on repetitive behaviors in Parkinson's disease
Authors: Friedman Joseph H.
Published: February 2005

A reply to this letter has been published in Movement DisordersMov Disord (2005) 20 (4) 509.
RNA in the nucleus of a motile plant spermatozoid: Characterization by enzyme‐gold cytochemistry and in situ hybridization
Authors: Rejon Emmanuelle , Bajon Catherine , Blaize Annie , Robert Daniel
Published: February 2005

To investigate the decrease of transcription and to determine the localization of RNA molecules during spermiogenesis in the fern Scolopendrium, we have used the enzyme‐gold electron microscope method. During cell differentiation, the labeling decreases over the cytoplasm and increases over the nucleus. In the latter, the nucleolus disappears at the beginning of differentiation and the gold particles that were at first situated over the dispersed chromatin are then located over the condensed chromatin. In mature gametes, gold particles linked to RNA are abundant over the dense and homogeneous nucleus and sparse over the cytoplasm. By using in situ hybridization of RNA‐poly(A) tails with radioactive poly(U) probes, we have shown that these RNA molecules are mRNA; they are located in the condensed chromatin of the mature sperm nucleus.
Chromatin arrangement in mouse sperm nuclei: An ultrastructural cytochemical study
Authors: Biggiogera M.
Published: February 2005

The arrangement of mouse sperm nuclei chromatin and, in particular, of DNA has been studied by electron microscopic cytochemistry. It had been previously shown that, after a Feulgen‐type reaction using an osmium ammine complex (OAC), the OAC‐stained DNA was distributed in a spotted pattern in the nucleus (Biggiogera: Basic Appl Histochem 30:501–504, 1986). The present chapter shows that this pattern is characteristic of mouse spermatozoa from testis to vas deferens, with the exception of some testicular spermatozoa, in which DNA was homogeneously stained. DNase digestion of thin‐sectioned nuclei resulted in a distribution of residual material complementary to the pattern of the unstained zones after the OAC reaction. These findings are discussed considering the role of –S–S– crosslinks, characteristics of this extremely condensed chromatin, in limiting the availability of DNA to acid hydrolysis.
Mouse zygotes injected with mitochondria develop normally but the exogenous mitochondria are not detectable in the progeny
Authors: Ebert Karl M. , Alcivar Acacia , Liem Hetty , Goggins Rick , Hecht Norman B.
Published: February 2005

A microinjection procedure to introduce “paternal” mitochondria from a source other than spermatozoa into fertilized mouse eggs is described. When a mitochondrial suspension isolated from the testes or liver of Mus molossinus mice was microinjected into fertilized eggs of CD1 mice, the microinjected zygotes survived, developed normally, and offspring were produced. Mus molossinus mitochondrial DNA can be distinguished from CD1 mitochondrial DNA by Southern blot analyses using restriction enzymes such as Eco R1, Xba 1, or Spe 1. Although up to 120 viable mitochondria were injected, no exogenous mitochondrial DNA was detected in fetal samples or in the brain, liver, heart, testis, or ovary of the mature progeny. Under the experimental conditions used, similar results were obtained when mitochondria from the testes of New Zealand black mice or from testes of Syrian hamsters were microinjected into fertilized CD1 mouse eggs. Failure to detect the exogenous mitochondrial DNA under our assay conditions suggests that microinjected mitochondria from testis or liver did not selectively replicate during embryonic development. The “foreign” mitochondria appear to have the same fate during early embryogenesis as the mitochondria of the spermatozoon.
Selection of mouse preimplantation embryos carrying exogenous DNA by polymerase chain reaction
Authors: Ninomiya Takashi , Hoshi Masaki , Mizuno Atsuko , Nagao Masaya , Yuki Atsushi
Published: February 2005

A polymerase chain reaction (PCR) system to detect transgenes in mouse preimplantation embryos was employed so that transgenic embryos could be selected before they were transferred to recipient mice. The selection system involves bisection of morulae, selection of the half‐morulae containing target sequences within 7 hr, and culture and transfer of the sister half‐morulae. PCR analysis of morulae derived from transgenic mice confirmed that the PCR system was reliable. However, five of 41 implanted embryos derived from PCR‐positive morulae did not contain the transgenes. Also, one of 28 implanted embryos from PCR‐negative morulae were transgenic. The selection system was applied to fertilized mouse eggs into which pSV2‐gpt‐gE1A DNA was injected. The injected DNA was detected in 30 of 84 morulae derived from the microinjected eggs. All seven implanted embryos developed from PCR‐negative morulae had no detectable amount of transgenes, and one of two successfully implanted embryos from PCR‐positive morulae was transgenic.
Qualitative changes in protein synthesis associated with polyspermic fertilization of human eggs
Authors: Dionne P. , Duchesne C. , Sullivan R.
Published: February 2005

To investigate the early molecular events in human oocytes that are triggered by fertilization, the authors examined the pattern of polypeptides synthesized by unfertilized and dispermic embryos obtained through an in vitro fertilization and embryo transfer (IVF‐ET) program. Compared with unfertilized oocytes of the same postovulatory age, the de novo protein synthesis in tripronuclear dispermic zygotes (21 hours postinsemination) was characterized by the appearance of three novel protein bands with molecular weights of 41.2, 35.3, and 26.0 kD. Concomitant with these changes, these zygotes showed the disappearance of bands at 54.0, 36.5, and 28.0 kD, along with the decreased synthesis of a protein band at 42.5 kD. Although 24% of the aged unfertilized oocytes exhibited bands corresponding to 41.2 and 35.3 kD, the 26.0 kD protein is restricted to the tripronuclear embryos. The significance of these results is discussed in relation to the use of polyspermic human oocytes as a model for the study of the early molecular events triggered by fertilization.
Spatial patterns of gene expression in preimplantation mouse embryos
Authors: Nisson Paul E. , Francis Susan , Crain William R.
Published: February 2005

The distribution of total polyadenylated RNA and mRNAs from the β‐actin, fibronectin, and cytokeratin Endo A genes was examined in preimplantation mouse embryos using in situ hybridization of riboprobes to RNA in sections of embryos. Polyadenylated RNA was found in the cytoplasm of all cells of blastocyst‐stage embryos, whereas the specific mRNAs displayed three distinct patterns of expression: uniform throughout the embryo (β‐actin), enriched in the inner cell mass (fibronectin), and enriched in the trophectoderm (Endo A). In eight‐cell embryos, the polyadenylated RNA was more concentrated in nuclei than in the cytoplasm (as noted previously), although this was not the case in blastocysts, nor was it true for the specific mRNAs that were examined. These experiments demonstrate that there is localized gene expression in the early mouse embryo, which correlates with the formation of the trophectoderm and the inner cell mass.
Gene transfer, expression and inheritance of PRSV‐rainbow trout‐GH cDNA in the common carp, Cyprinus carpio (Linnaeus)
Authors: Zhang Peijung , Hayat Mohammad , Joyce Christopher , Gonzalez‐Villaseñor Lucia Irene , Lin C. M. , Dunham Rex A. , Chen Thomas T. , Powers Dennis A.
Published: February 2005

A recombinant plasmid containing the Rous sarcoma virus‐long terminal repeat (RSV‐LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV‐LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV‐rtGH‐cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one‐cell stage were (P < 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non‐transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P < 0.05) than their non‐transgenic siblings.
Stage‐Specific expression of the lactate dehydrogenase‐X gene in adult and developing mouse testes
Authors: Jen J. , Deschepper C. F. , Shackleford G. M. , Lee C. Y. G. , Lau Y.‐F. C.
Published: February 2005

Lactate dehydrogenase‐X (LDH‐X), a glycolytic enzyme found only in mammalian testes and spermatozoa, is encoded by a single gene (Ldh‐x) in the mouse haploid genome. Several studies have demonstrated that LDH‐X is associated with germ cells at specific stages of development. We have examined the expression of the Ldh‐x gene during mouse spermatogenesis and testis maturation using in situ mRNA hybridization and immunocytochemistry. The results showed that transcription and translation of the Ldh‐x gene are initiated at the pachytene stage of germ cell differentiation. However, although the amount of LDH‐X protein increased as the germ cells progressed to maturation, its mRNA level was greatly decreased. These observations were confirmed by Northern analysis of total RNA derived from fractionated spermatogenic cells and developing testes. Furthermore, Northern studies also indicated two sizes of Ldh‐x transcripts among different populations of spermatogenic cells in mature mouse testis.
Collection and quality of rhesus monkey semen
Authors: Lanzendorf S. E. , Gliessman P. M. , Archibong A. E. , Alexander M. , Wolf D. P.
Published: February 2005

Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electrcejaculation: (1) a constant‐voltage method where stimulus current is a variable and (2) a constant‐current method where stimulus current is operator‐controlled. The constant‐current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The intluence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were determental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra‐ and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.
A comparison of fertilization envelope development in three species of Strongylocentrotus (S. Franciscanus, S. Droebachiensis, and S. purpuratus)
Authors: Carroll Edward J. , Cohen Jeffrey S.
Published: February 2005

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (Pl), and final structuralization was observed by 40 minutes Pl. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes Pl. The S. droebachiensis FE retained microvillar casts, assumed its definitive from by 3 minutes Pl, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes Pl were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against exttracted FEs and the FE extract from S. purpuratus were immunologically cross‐reactive (using an enzyme‐linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross‐reactive polypeptides, respectively. Most of the cross‐reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.
Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus
Authors: Arceci Robert J. , Baas Frank , Raponi Romeo , Horwitz Susan Band , Housman David , Croop James M.
Published: February 2005

The multidrug resistance (mdr) gene family has been shown to encode a membrane glycoprotein, termed the P‐glycoprotein, which functions as a drug efflux pump with broad substrate specificity. This multigene family is expressed in a tissue‐specific fashion in a wide variety of normal and neoplastic tissues. The regulation of mdr gene expression in normal tissues is not understood. We have recently shown that mdr mRNA and the P‐glycoprotein increases dramatically in the secretory luminal and glandular epithelium of the gravid murine uterus. This observation has suggested that mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy. This report now demonstrates that mdr mRNA and P‐glycoprotein are induced at high levels in the uterine secretory epithelium by the combination of estrogen and progesterone, the major steroid hormones of pregnancy. This regulation of mdr gene expression in the uterus does not require any other contribution from the fetus or placenta. The data indicate that this gene locus is hormonally responsive to estrogen and progesterone in the uterine secretory epithelium, suggesting an important and physiologically regulated role during pregnancy.
First cell cycle of zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo
Authors: Boerjan M. L. , De Boer P.
Published: February 2005

This paper describes an analysis of the first cell cycle of mouse oocytes aged postovulation and fertilized in vivo. For this purpose, we developed a procedure for inducing ovulation in vivo that allows accurate timing of ovulation. The method is based on a luteinizing hormone (LH)‐releasing hormone (LHRH) administration at prcestrus. This ovulation procedure had no detectable effect on the rate of ovulation or postimplantation embryonic death. We used this method of ovulation induction in an analysis of the separate stages of the first cell cycle of in vivo fertilized postovulation aged oocytes. All stages assessed were shorter in aged oocytes (12 hr postovulation) than in zygotes from unaged oocytes (1 hr postovulation): (1) the time interval between insemination and penetration of the aged oocytes was 1.5 hr shorter than the time interval of the unaged oocytes; (2) pronuclear formation in the fertilized aged oocytes was somewhat quicker than pronuclear formation in fertilized unaged oocytes; (3) in zygotes from aged oocytes, the time between formation of pronuclei and the pronuclear membrane breakdown was 1 hr shorter than in zygotes from unaged oocytes; (4) the first cleavage division was 3 hr advanced in zygotes from aged oocytes compared with the moment of the first cleavage division in zygotes from unaged oocytes. We also determined the glutathione (GSH) content of unaged and aged oocytes to investigate a possible relationship between the rate of pronuclear formation and GSH. The level of GSH was two times lower in oocytes aged postovulation for 12 hr than in unaged oocytes. The level of GSH in fertilized, unaged oocytes was half that in unfertilized, unaged oocytes; this decline was not observed after fertilization of aged oocytes, however. In summary, we developed a procedure that allows in vivo fertilization at defined points of time after LHRH‐induced ovulation. Under the conditions used in this procedure, we could detect several differences between zygotes derived from unaged and postovulation aged oocytes.
Changes in the mitochondrial calcium influx and efflux properties are responsible for the decline in sperm calcium during epididymal maturation
Authors: Vijayaraghavan S. , Hoskins D. D.
Published: February 2005

This study was undertaken to determine the role of calcium ion, a key regulator of the intensity and form of motility in mature demembranated sperm, in the development of motility during passage through the bovine epididymis. Cellular calcium levels in bovine caput and cauda epididymal spermatozoa were measured with three different techniques. 45Ca2+ uptake measurements revealed that net calcium uptake and Ca2+‐Ca2+ exchange in caput spermatozoa were about 2 to 3 times higher than in caudal spermatozoa. Intracellular free calcium determination with the calcium fluorophore Fura 2 showed that the levels were 6 times higher in caput spermatozoa. The values for caput and caudal sperm were 875±55 nM (n = 15) and 155±6 nM (n = 24), respectively. Total cellular calcium levels quantitated by atomic absorption were 626±30 (n = 48) and 304±19 (n = 46) ng/108 sperm in caput and caudal epididymal sperm, respectively. At least one of the reasons for the high calcium content of caput epididymal sperm is the result of a higher rate and extent of mitochondrial calcium accumulation in caput compared to caudal sperm. Mitochondrial calcium uptake rates measured in digitonin permeabilized cells revealed uptake rates 2‐ to 3‐fold higher in caput compared to caudal sperm. However, mitochondrial calcium efflux rates were identical in caput and caudal epididymal sperm. The efflux rates in both cell types were unaffected by external sodium levels but were found to be proportional to pH. Alkalinization or acidification of internal pH of intact sperm resulted in a corresponding lowering or elevation of cytoplasmic free calcium levels. We propose that external calcium has access to sperm only via the mitochondria (Vijayaraghavan and Hoskins: Cell Calcium 10:241–253, 1989) and that this mitochondrial calcium is subsequently redistributed into the cytoplasmic space as a function of the internal pH. We document for the first time that changes in mitochondrial calcium handling properties are important in epididymal sperm maturation and suggest that the acquisition of sperm motility in the epididymis could be related to these changes in sperm calcium handling properties.
Effects of ultrasound on DNA and RNA synthesis in preimplantation mouse embryos
Authors: Heyner S. , Abraham V. , Wikarczuk M. L. , Ziskin M. C.
Published: February 2005

Ultrasound is used extensively to monitor the growth of ovarian follicles in in vitro feritilization and embryo transfer (IVF‐ET) programs, as well as to follow the progress of early pregnancy. There have been scattered reports in the literature that exposure to ultrasound may have an adverse effect on reproduction in the rat (Bologne et al: CR Soc Biol 177:381–387, 1983; Demoulin et al: Ann Ny Acad Sci 442:146–152, 1985), and also in humans (Demoulin et al: Ann NY Acad Sci 442:146–152, 1985). We report here that diagnostic levels of pulsed ultrasound did not affect either the number of embryos produced, or the ability to incorporate labelled precursors into DNA and RNA, respectively. Measurements of temperature elevation of ovaries exposed to ultrasound showed that neither controls nor experimental tissue exhibited temperature elevation greater than 1°C.
Assessment of the human sperm acrosome reaction using concanavalin a lectin
Authors: Holden Carol A. , Hyne Ross V. , Sathananthan A. H. , Trounson Alan O.
Published: February 2005

A method for assessment of the human sperm acrosome reaction is reported using fluorescein isothiocyanate (FITC)‐conjugated Concanavalin A (ConA). The technique involved labelling prefixed spermatozoa, where only those spermatozoa that showed a complete loss of the acrosome bound FITC‐ConA to the acrosomal region. Competitive sugar binding studies demonstrated that binding of ConA lectin to the acrosomal area of human spermatozoa was inhibited in the presence of 0.2 M D‐mannose. Staining with the supravital stain Hoechst 33258 (H258) concomitantly with FITC‐ConA allowed determination of only those spermatozoa that had undergone a true and not degenerative acrosomal loss. Incubation of human spermatozoa with 0, 1, 5, and 25 μM calcium ionophore, A23187, for 60 min demonstrated that changes in acrosomal status due to the different treatment protocols may be determined by the dual‐staining method. Electron microscopy studies revealed that gold‐conjugated ConA bound specifically to the surface of the inner acrosomal membrane of acrosome‐reacted spermatozoa. A significant correlation (r = +.97) between transmission electron microscopy (TEM) and FITC‐ConA labelling methods of acrosomal status assessment was achieved. The simple ConA labelling procedure reported here therefore provides a reliabale method for quantitation of the physiological acrosome reaction of a population of human spermatozoa.
Spermatogenesis‐related change in the synthesis of the creatine kinase B‐type and M‐type isoforms in human spermatozoa
Authors: Huszar Gabor , Vigue Lynne
Published: February 2005

We have demonstrated earlier that the per sperm creatine‐N‐phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B‐CK and M‐CK isoforms during cellular differentation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 ± 3.7 vs. 8.8 ± 1.3 million sperm/ml; all values expressed as mean ± SEM), the relative concentrations of the M‐CK isoform (M‐CK / M‐CK+B‐CK) were 27.2% ± 2.1% vs. 6.7% ± 0.9% (P < 0.001). The per sperm CK activities showed comparable differences (0.21 ± 0.02 vs. 0.89 ± 0.1 CK IU/100 million sperm; P < 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M‐CK concentrations (R = 0.69, P < 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M‐CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M‐CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M‐CK, the muscle‐sperm MB‐CK hybrid has formed. Thus in sperm and muscle the M‐CK isoforms are structurally different, whereas the B‐CKs are apparently homologous. We suggest that the relative concentrations of sperm M‐CK isoform is a biochemical marker of cellular differentiation that may also predict the functional intetrity, such as fertilization potential, of spermatozoa.