bluuebunny/crossref_metadata_2025 huggingface dataset:
Plate Fixation of Prostheses after Segmental Resection for Bone Tumours
Authors: Coathup Melanie J. , Cobb Justin P. , Walker Peter S. , Blunn Gordon W.
Published: February 2005

This study investigated the concept of using plates to attach endoprostheses to bone after segmental resection for bone tumours in an animal model. Titanium alloy plates integrated with the prosthesis and coated with hydroxyapatite were attached to bone by screws. This type of uncemented fixation relied on the induction of periosteal bone formation into and around the plates to secure the implant to bone. Tow, three, and six‐slotted plate designs were investigated. On retrieval, each plate was securely fixed by new bone. Bone apposition on the external surface of the plates occurred through a combination of periosteal bone production, invasion of bone through slots in the plate, and bone growth over the ends of the plates. Most plates became incorporated into a remodelled cortex. Higher bone turnover rates (μm day−1) were seen in bone in the slots of the plate compared with normal cortical bone turnover (p < 0.05). Significantly higher rates of turnover were measured beneath slotted parts of the plates compared with regions below the unslotted parts (p < 0.05). The cross‐sectional area of bone surrounding the six‐plate implant design was significantly higher than that of the three‐plate (p < 0.05) and two‐plate (p < 0.05) designs. In addition, significantly more bone formed adjacent to the six‐plated implant design compared with that in the contralateral limb (p = 0.002). However, no significant difference was found when the total cortical area around the three‐plated design was compared with that of the contralateral limb (p = 0.63). In contrast, significantly less bone was measured adjacent to the two‐plate design than in the untreated limb (p = 0.001). Image analysis also demonstrated increased cortical porosity adjacent to the six‐plate design compared with the three‐plate (p = 0.004) and two‐plate (p < 0.05) designs. Finite element analysis demonstrated that the six and three‐plate designs increased the second moment of area compared with that in the left tibia (p = 0.003 and 0.066, respectively). However, the attachment of the more flexible two‐plate design did not significantly increase the second moment of area compared with that in the contralateral limb (p = 0.235). It was concluded that due to both mechanical and biological effects, the hydroxyapatite‐coated plate designs generated new bone that enhanced fixation and encouraged plate integration into the load‐bearing structure of the cortex. This method of fixation may be an alternative to the use of intramedullary cemented stems in patients requiring bone tumour implants and may be the only way to preserve the joint in difficult cases where only short segments of bone remain.
Enhanced proliferation and differentiation of human articular chondrocytes when seeded at low cell densities in alginate In Vitro
Authors: Gagne T. A. , Chappell‐Afonso K. , Johnson J. L. , McPherson J. M. , Oldham C. A. , Tubo R. A. , Vaccaro C. , Vasios G. W.
Published: February 2005

Dedifferentiated human articular chondrocytes exhibited a wide variation in their capacity to proliferate and redifferentiate in an alginate suspension culture system. The greatest extent of proliferation and redifferentiation was seen to be dependent on the formation of clonal populations of chondrocytes and correlated mversely with the initial cell seeding density. Redifferentiating chondrocytes seeded at low density (1 × 104 cells/ml alginate) compared with chondrocytes that were seeded at high density (1 × 106 cells/ml alginate) showed a nearly 3‐fold higher median increase in cell number, a 19‐fold greater level of type‐II collagen mRNA expression, a 4‐fold greater level of aggrecan mRNA expression, and a 6‐fold greater level of sulfated glycosaminoglycan deposition at 4 weeks of culture. Matrix molecules from low‐density cultures were assembled into chondrocyte‐encapsulated, spherical extracellular matrices that were readily visualized in sections from 12‐week cultures stained with antibodies against types I and II collagen and aggrecan. Ultrastructural analysis of 12‐week low‐density cultures confirmed the presence of thin collagen fibrils throughout the matrix.
Extracellular collagen regulates expression of transforming growth factor‐β1 gene
Authors: Qi Wen‐Ning , Scully Sean P.
Published: February 2005

A complex interrelationship exists between the extracellular matrix and cytokine signaling in articular chondrocytes. We sought to determine whether the extracellular matrix serves as a regulatory component of transforming growth factor‐β1 expression. Bovine articular chondrocytes were isolated and resuspended in alginate, yielding final extracellular protein concentrations of 0 to 1.5% (wt/vol) for type‐II or type‐I collagen. Cultures were maintained for 7 days in the presence or absence of transforming growth factor‐β1‐supplemented medium (10 ng/ml). The amount of transforming growth factor‐β1 mRNA was examined with quantitative competitive reverse transcription‐polymerase chain reaction analysis. The results indicate that exogenous transforming growth factor‐β1 stimulates endogenous transforming growth factor‐β1 mRNA expression approximately 8‐fold. This effect depended on the concentration of extracellular type‐II collagen. As the concentration of extracellular type‐II collagen is increased, the expression of transforming growth factor‐β1 mRNA decreases in both basal and transforming growth factor‐β1‐stimulated cultures. Exogenous extracellular type‐I collagen also served to negatively modulate transforming growth factor‐β1 gene expression but with a different concentration profile. The results demonstrate that transforming growth factor‐β1 mRNA expression was upregulated by exogenous transforming growth factor‐β1 and was downregulated by extracellular type‐I and type‐II collagens. The profoundly different effects on transforming growth factor‐β1 expression by the two collagens are consistent with those reported for mammary epithelial cells and likely serve as a negative feedback mechanism to preserve tissue homeostasis.
Kinetics of oxidation of pantothenic acid by chloramine‐T in perchloric acid and in alkaline medium catalyzed by OsO4: A mechanistic approach
Authors: Puttaswamy  , Jagadeesh R. V.
Published: February 2005

Kinetics of oxidation of pantothenic acid (PA) by sodium N‐chloro‐p‐toluenesulfonamide or chloramine‐T (CAT) in the presence of HClO4 and NaOH (catalyzed by OsO4) has been investigated at 313 K. The stoichiometry and oxidation products are same in both media; however, their kinetic patterns were found to be different. In acid medium, the rate shows first‐order dependence on [CAT]o, fractional‐order dependence on [PA]o, and inverse fractional‐order on [H+]. In alkaline medium, the rate shows first‐order dependence each on [CAT]o and [PA]o and fractional‐order dependence on each of [OH−] and [OsO4]. Effects of added p‐toluenesulfonamide and halide ions, varying ionic strength, and dielectric constant of medium as well as solvent isotope on the rate of reaction have been investigated. Activation parameters were evaluated, and the reaction constants involved in the mechanisms have been computed. The proposed mechanisms and the derived rate laws are consistent with the observed kinetics. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 201–210, 2005
A statistical/collisional approach to rates of bimolecular reactions at low pressure
Authors: Canepa Carlo
Published: February 2005

An expression for the rate constant of gas‐phase bimolecular reactions at low pressure is derived in the framework of collisional theory. The key feature of the proposed model is the calculation of the energy‐dependent rate constant in terms of the collisional cross section and the probability of reaction, expressed as the ratio of the volume in phase space that leads to product over the total volume. The contribution of the internal energy of the reacting fragments is taken into account, as well as the relative translational energy. The resulting formulation is able to account for both negative and positive temperature dependences of the rate constants of neutral and charged species. The dependence of temperature of the bimolecular rate constant is given both for reactions with and without potential energy barriers. The performance of the proposed model is tested against experimental rate constant for three well‐studied reactions by fitting the parameters of the model to experimental data at various temperatures. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 233–242, 2005
Kinetics and mechanism of ligand exchange of coordinated cyanide in hexacyanoferrate(II) by nitroso‐R‐salt in the presence of mercury(II) as a catalyst
Authors: Naik R. M. , Sarkar Joy , Chaturvedi D. D.
Published: February 2005

The kinetics of Hg(II)‐catalyzed reaction between hexacyanoferrate(II) and nitroso‐R‐salt has been followed spectrophotometrically by monitoring the increase in absorbance at 720 nm, the λmax of green complex, [Fe(CN)5 N‐R‐salt]3− as a function of pH, ionic strength, temperature, concentration of reactants, and the catalyst. In this reaction, the coordinated cyanide ion in hexacyanoferrate(II) gets replaced by incoming N‐R‐salt under the following specified reaction conditions: temperature = 25 ± 0.1°C, pH = 6.5 ± 0.2, and I = 0.1 M (KNO3). The stoichiometry of the complex has been established as 1:1 by mole ratio method. The rate of catalyzed reaction is slow at low pH values and then increases with pH and attains a maximum value between 6.5 and 6.7. The rate finally falls again at higher pH values due to nonavailability of [H+] ions needed to regenerate the catalytic species. The rate of reaction increases initially with [N‐R‐salt] and attains a maximum value and then levels off at higher [N‐R‐salt]. The rate of reaction shows a variable order dependence in [Fe(CN)64−] ranging from unity at lower concentration to 0.1 at higher concentrations. The effect of [Hg2+] on the reaction rate shows a complex behavior and the same has been explained in detail. The activation parameters for the catalyzed reactions have been evaluated. A most plausible mechanistic scheme has been proposed based on the experimental observations. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 222–232, 2005
Kinetics and mechanism of the C2H5O2 + NO reaction: A temperature and pressure dependence study using chemical ionization mass spectrometry
Authors: Bardwell Max W. , Bacak Asan , Raventós M. Teresa , Percival Carl J. , Sanchez‐Reyna Gabriela , Shallcross Dudley E.
Published: February 2005

The overall rate coefficient (k1) for the reaction of C2H5O2 + NO has been measured using the turbulent flow CIMS technique. The temperature dependence of the rate coefficient was investigated between 203 and 298 K. Across the temperature range, the experimentally determined rate coefficients showed good agreement with previous studies and were fitted using an Arrhenius type analysis to yield the expression k1 = (1.75) × 10−12 exp[(462 ± 19)/T] cm3 molecule−1 s−1. Experiments were carried out in the pressure range of 100–200 Torr within the stated temperature range, where the rate coefficients were shown to be invariant with pressure. The branching ratio of the reaction was also assessed as a function of temperature and was found to proceed 100 ± 5% via the C2H5O + NO2 reactive channel. This work represents the first temperature and pressure study over which the branching ratio has been studied. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 253–260, 2005
Improved Adhesion of UV Treated EVA Copolymer by Adding Small Amounts of Fillers
Authors: Landete‐Ruiz María Dolores , Martín‐Martínez José Miguel
Published: February 2005

The influence of adding small amounts of calcium carbonate or sepiolite fillers on the adhesion properties of UV surface‐treated ethylene vinyl acetate copolymer (EVA) was studied. In this paper, it has been demonstrated that the addition of 3‐7 wt% filler enhanced the extent of the surface modifications (wettability, surface chemistry, roughness) and increased the adhesion of EVA. These effects were more marked when 7 wt% sepiolite filler was added to EVA, likely due to its higher aspect ratio.
Effect of Unmodified and Surface Treated Fumed Silica on the Polymerization of HEMA
Authors: Tertykh Valentin , Bolbukh Yulia , Yanishpolskii Victor
Published: February 2005

Thermal polymerization of 2‐hydroxyethylmethacrylate (HEMA) and radical polymerization initiated by an acrylamid complex of cobalt nitrate in the presence of unmodified and surface treated silicas have been investigated. It has been found that the introduction of fillers, especially silicas with grafted silicon hydride groups, makes an essential effect on the rate and degree of the HEMA polymerization.
The Effect of Glass Fibre and a Phosphorus‐Containing Flame Retardant on the Flammability of Recycled PET
Authors: Maspoch Maria Lluïsa , Ferrando Haritz E. , Vega Diego , Gordillo Antonio , Velasco José Ignacio , Martínez Antonio B.
Published: February 2005

The availability of scrap poly(ethylene terephthalate) (PET) from post‐consumer bottles is increasing as the post‐consumer collecting systems are becoming more usual in daily life. PET is well known as a high‐performance engineering thermoplastic because of its good thermal stability, chemical resistance, and excellent mechanical properties. Many efforts have been carried out to use this material in housings of electronic applications. However, the flammability of PET is a shortcoming in some of these applications.

In this study, our attempt is to incorporate a non‐halogenated flame retardant, in form of a phosphorus‐containing compound, together with a commercial glass fibre grade to achieve UL94 test V‐0 rating for PET. An investigation of thermal stability and flammability (HDT, UL94 V‐test) and mechanical (tensile, flexural and impact tests) properties of glass fibre filled PET samples is reported as a function of fraction of flame retardant.

This work shows the influence of the filler content and the interfacial filler/matrix adhesion on the flame retardant and the mechanical properties.
Reply: Levodopa in the treatment of Parkinson's disease: Current controversies
Authors: Olanow C. Warren , Agid Yves , Mizuno Yoshi
Published: February 2005

The original articles to which this Letter refers have published in Movement Disorders: Current Controversies: Levodopa in the Treatment of Parkinson's Disease, by Sharma, Vassallo, and Ross and Levodopa in the Treatment of Parkinson's Disease: Current Controversies, by Gerlach, Reichmann, and Riederer.
Current controversies: Levodopa in the treatment of Parkinson's disease
Authors: Sharma Jagdish C. , Vassallo M. , Ross I.N.
Published: February 2005

Companion letters have been published in Movement Disorders: Levodopa in the Treatment of Parkinson's Disease: Current Controversies, by Gerlach, Reichmann, and Riederer and Reply: Levodopa in the Treatment of Parkinson's Disease, by Olanow, Agid, and Mizuno.
Reply: Repetitive behaviors in Parkinson's disease
Authors: Voon Valerie
Published: February 2005

The original letter to which this reply refers was published in Movement DisordersMov Disord (2005) 20 (4) 509.
Microtubule formation from maternal tubulins during sea urchin embryogenesis: Measurement of soluble and insoluble tubulin pools
Authors: Gong Zhiyuan , Brandhorst Bruce P.
Published: February 2005

The mass of tubulin protein in developing embryos of the sea urchin Lytechinus pictus was measured using a radiodilution immunoassay based on densitometric analysis of immunoprecipitated tubulins resolved electrophoretically. The tubulins constitute an average of 360 ± 35 pg per egg, or 0.66% of the total protein, and there is no significant change in their concentration during embryogenesis. The masses of soluble and polymerized tubulins were measured for extracts prepared under conditions that stabilize microtubules. In eggs, a maximum of 14% of the tubulin is insoluble, and this increases throughout embryogenesis to 67% at pluteus stage (72 hr). The concentration of tubulin in eggs is at least 500 μg/ml, well above the critical concentration for tubulin assembly in vitro, yet microtubules have not been observed in eggs. The mass of newly synthesized tubulin, estimated from the mass of tubulin mRNA per embryo, accounts for a small fraction of the total tubulin by the end of gastrulation but for over half of the tubulin by the 72‐hr pluteus stage. These observations are consistent with a model in which the declining level of unpolymerized tubulin controls the stability of tubulin mRNA, providing an autogenous regulation of the ontogenetic pattern of tubulin synthesis during sea urchin embryogenesis (Gong and Brandhorst, Development 102: 31–43).
Identification of specific gene sequences in preimplantation embryos by genomic amplification: Detection of a transgene
Authors: King Donna , Wall Robert J.
Published: February 2005

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous single copy gene or an integrated foreign gene. The endogenous single‐copy gene encoding the β chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance where bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunify to study genetics in early development.
Nucleologenesis and the onset of transcription in the eight‐cell bovine embryo: Fine‐structural autoradiographic study
Authors: Kopečný V. , Fléchon J. E. , Camous Sylvaine , Fulka J.
Published: February 2005

Eight‐cell cow embryos were isolated and cultured in vitro in a medium enriched with 200 μCi of [5‐3H]uridine for 20 min. Epon ultrathin sections of the embryos were investigated for the nucleolar morphology and for the appearance and localization of the sites of [5‐3H]uridine incorporation by means of electron microscopic autoradiography. In addition to this, a general pattern of replicated embryonal DNA distribution was revealed by [methyl‐3H]thymidine incorporation and light microscopic autoradiography.

The essential phases of the transformation of the small nucleous precursor body (NPB) into a vast, functionally fully active nucleolus, characterized by typical nucleolar substructural components, are taking place within the eight‐cell stage. This process differed in its morphology from the nucleologenetic process in early embryogenesis of other mammals, especially of that in the mouse.

The first sign of NPB, transformation was the appearance of a large central vacuole followed later on by perinucleolar chromatin penetration into NPB, documented by both morphology and [3H]thymidine autoradiography. In some cases, concentration of dense fibrillar material forming clumps or stalks was seen in the central vacuole.

The following rapid nucleolar development was characterized by the formation of secondary vacuoles concomitant with the onset of [5‐3H]uridine incorporation into the dense fibrillar component and with the appearance of the first granules in the otherwise fibrillar structure of the nucleolus. During the late eight‐cell stage, the still‐rounded nucleolus developed features of a reticulated nucleolus known from somatic cells intensively synthesizing rRNA: a dense fibrillar component with associated labeling encircling fibrillar centers and a well‐developed granular component. The labeled dense fibrillar component was observed mostly in the central area of the nucleolus; early embryonic NPB dense fibrous material not involved in transcription was disappearing rapidly. At the transition to the 16‐cell stage the nucleoli lost their rounded shape because of the accumulation of a large amount of granular component, and they occupied a considerable part of the nucleus. In conclusion, the appearance of the nucleolar vacuole in eight‐cell cow embryo is the starting point for following morphogenetic events linked with the onset of transcription.
Posttranscriptional control of human homeobox gene expression in induced NTERA‐2 embryonal carcinoma cells
Authors: Simeone Antonio , Acampora Dario , D'Esposito Maurizio , Faiella Antonio , Pannese Maria , Scotto Luigi , Montanucci Manuela , D'Alessandro Gaetana , Mavilio Fulvio , Boncinelli Edoardo
Published: February 2005

We have studied the expression of four human homeobox genes representative of four different clusters (i.e., HOX‐1, HOX‐2, HOX‐3 and HOX‐5) in the embryonal carcinoma (EC) cell line NT2/D1. Following treatment with retinoic acid (RA), these cells differentiate into several cell types, including neurons, and steadily accumulate polyadenylated transcripts derived from the genes in a period ranging from 18 hr to 14 days of RA treatment. The sizes of major transcripts in differentiated EC cells coincide with those previously detected by the same probes in human embryos. Nuclear run‐on transcriptional analysis showed no difference in the transcription rate of the four homeobox genes in differentiated vs. undifferentiated EC cells. Inhibition of protein synthesis by 5–18 hr of treatment of undifferentiated cells with cycloeximide causes accumulation of some homeobox transcripts at levels comparable to those observed after 18 hr of RA induction, although it does not cause superinduction in fully differentiated cells. These data suggest that the activation of homeobox gene expression in RA‐induced EC cells is controlled, at least in part, by posttranscriptional mechanisms.
Production of transgenic sheep with growth‐regulating genes
Authors: Rexroad Caird E. , Hammer Robert E. , Bolt Douglas J. , Mayo Kelly E. , Frohman Lawrence A. , Palmiter Richard D. , Brinster Ralph L.
Published: February 2005

Pronuclei of fertilized sheep ova were injected with fusion genes consisting of the mouse metallothionein‐I promotor/regulator ligated to either the structural gene for bovine growth hormone (mMTbGH) or to a minigene for human growth hormone‐releasing factor (mMThGRF). From a total of 842 sheep ova injected with mMTbGH and transferred into recipient ewes, 47 lambs were born. Two of the lambs were transgenic with mMTbGH, and both had bGH mRNA present in liver, kidney, and gut. In one lamb, plasma growth hormone was as high as 700 ng/ml. From a total of 435 sheep ova injected with mMThGRF and transferred to recipients, 54 lambs were born and 9 fetuses were collected. Nine of the 63 had integrated the mMThGRF gene. One of the nine had high concentrations of immunoassayable hGRF in its plasma and high variable plasma concentrations of ovine growth hormone. The lamb that expressed the hGRF gene did not release GH in response to an hGRF challenge. Four of five fetal offspring of a nonexpressing mMThGRF transgenic ram also contained the mMThGRF gene and, like the sire, failed to express the gene as determined by either liver hGRF mRNA or by plasma hGRF. Growth of the single transgenic lamb expressing hGRF was similar to control lambs. These studies demonstrate efficient introduction of genes into the sheep genome and indicate that transgenes are expressed and heritable.
DNA sequence and pattern of expression of the sea urchin (Paracentrotus lividus) α‐tubulin genes
Authors: Gianguzza F. , Bernardo M. G. Di , Sollazzo M. , Palla F. , Ciaccio M. , Carra E. , Spinelli G.
Published: February 2005

To study the molecular aspects of the regulation of transcription of a multigene family, we have isolated and sequenced cDNA and genomic clones coding for the α‐tubulin of the sea urchin Paracentrotus lividus. Two cDNA clones, Pα 10 and Pα 4, contain respectively the coding information for 391 C‐terminal and for 338 N‐terminal amino acids of the 452 residues that constitute the complete protein. They show silent nucleotide substitutions only, suggesting that Pα 10 and Pα 4 represent the cloned copies of two allelic gene transcripts, which encode for two α‐tubulin isoforms with identical amino acid sequence in the region of the overlap. The comparison of the predicted amino acid sequence of the composite Pα 4–10 and of the mouse M α‐6 (Villasante et al., Mol. Cell Biol 1986; 6:2409–2419) reveals a conservation of 97% between the two polypeptides. By RNA blotting hybridization six major α‐tubulin transcripts were identified. Two, of 3.5 kb and 2.0 kb, are expressed in the unfertilized eggs and during early cleavage. The other two maternal mRNAs, of 2.4 kb and 1.8 kb, are expressed in both early and late cleavage embryos, but in the intestine the 1.8 kb RNA, which specifically reacted with the 3′ specific probe of the Pα 10 cDNA, is the only transcript detected. Finally, the 1.5 kb and 1.9 kb mRNAs represent the transcription of stage‐ and tissue‐specific genes, respectively. In fact, the former becomes detectable at blastula stage and accumulates during late development, whereas the latter is found in the testis only. The sequence data of the 3′ terminus of the α‐3 genomic clone suggests that it encodes for a divergent α‐tubulin, and it most probably corresponds to the testis‐specific gene.
Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis
Authors: Yelick Pamela C. , Kwon Yunhee Kim , Flynn James F. , Borzorgzadeh Adel , Kleene Kenneth C. , Hecht Norman B.
Published: February 2005

Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.
Histone gene expression during sea urchin spermatogenesis: An in situ hybridization study
Authors: Poccia Dominic , Lieber Toby , Childs Geoffrey
Published: February 2005

The expression of testis‐specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis‐specific histone genes (Sp H2B‐1 of Strongylocentrotus purpuratus and Sp H2B‐2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse‐labeled with 3H‐thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A‐β for S. purpuratus and H3‐1 for L. Pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis‐specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis‐specific gene expression is restricted to spermatogonia and premeiotic spermatid, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.
Transgenic offspring by transcaryotic implantation of transgenic ovaries into normal mice
Authors: Brem Gottfried , Baunack Eva , Müller Mathias , Winnacker Ernst‐Ludwig
Published: February 2005

Female transgenic mice may be unable to reproduce successfully if the product encoded by the transgene results in pathological changes or affects the fertility of the mouse. To approach this problem, we have produced chimaeras by transferring the ovaries of transgenic mice into normal mice of the same strain. Such chimaeras will be an ideal tool for investigating the interactions between transgenic ovaries and normal mice or vice versa. Here we show that, using this method, we were able to get large numbers of transgenic offspring even from founder transgenic female mice that were themselves infertile as a result of the overexpression of growth hormone genes. Although none of the ovary recipients were given immunosuppressant treatment, 60% of the recipients had biologically active ovaries over a mean period of about 100 days.
Proacrosin activation and acrosin release during the guinea pig acrosome reaction
Authors: Nuzzo Nancy A. , Anderson Robert A. , Zaneveld Lourens J. D.
Published: February 2005

The kinetics of proacrosin activation and release from guine pig spermatozoa during the nonsynchronous acrosome reaction were studied. Epididymal spermatozoa were incubated at 37°C in a defined medium (pH 7.8) containing 1.7 mM Ca2+. After 195 min, 78% of the motile spermatozoa had undergone the acrosome reaction as determined by light microscopy. Acrosin and proacrosin levels in the spermatozoa and medium were measured at the beginning of the incubation period. Most of the total acrosin activity (78%) was associated with the spermatozoa, of which greater than 90% was in the form of proacrosin. Proacrosin represented a small, stable fraction (23%) of the total acrosin in the medium; it did not activate to acrosin while in the medium. After 195 min, a decrease in sperm‐associated total acrosin (42%; p < 0.05) was accompanied by an increase in the total acrosin level in the medium (115%; P < 0.05). No change in the relative proacrosin content (percent of total acrosin) was evident in either medium or spermatozoa. Additional experiments quantified acrosin and proacrosin during the progression of the acrosome reaction. Both the loss of sperm‐associated total acrosin and the increase in total acrosin levels in the medium were highly correlated with the fraction of acrosome‐reacted spermatozoa (r = 0.954 and 0.922, respectively; P < 0.001). However, the rate of acrosin appearance in the medium was only 60% (P < 0.001) of the rate of acrosin loss from the spermatozoa. The fractional proacrosin content of spermatozoa (94%) and medium (31%) remained unchanged during the acrosome reaction (r = 0.15 and 0.30, respectively; P > 0.1). From these data, it was possible to estimate the total acrosin content of acrosome‐intact spermatozoa (434 mlU/107 spermatozoa), and the fractions of the total sperm acrosin levels that are (1) released as active acrosin (37%), (2) released as proacrosin (17%), (3) inactivated (36%), and (4) associated with the spermatozoa (10%) during the acrosome reaction. The data suggest that proacrosin activation occurs within the spermatozoa, prior to distruption of the acrosome; this finding is consistent with the contention that acrosin is an active component of the mechanism underlying the acrosome reaction.
Diadem/crater defects in spermatozoa from two related angus bulls
Authors: Larsen Rolf E. , Chenoweth Peter J.
Published: February 2005

The diadem/crater defect was studied over several months in two related 20‐month‐old Angus bulls. In bull 1, diadem/crater defects were present in 2–99% of ejaculated spermatozoa at various times during the evaluation period. In bull 2, affected cells varied from 20% to 94%, with other abnormalities (head and acrosome defects, coiled tails, proximal cytoplasmic droplets) also common. Single sire mating trials conducted over 26 days during an apparent recovery phase showed normal fertility (approximately 50% pregnancies per estrus exposed). Both resting and gonadotropin‐releasing hormone (GnRH)‐stimulated testosterone values were within nor‐mal limits. Histopathological evaluation of testes showed no obvious hypoplastic, inflammatory, or degenerative condition. Electron microscopy of ejaculated spermatozoa demonstrated the characteristic diadem pattern of craters in the equatorial region of the head. Many cells from bull 2 contained large craters in other regions of the nucleus. Electron microscopy of testicular tissue demonstrated nuclear invaginations lined by a single unit membrane in round spermatids. Lesions in elongated spermatids were more pronounced, with curling of the nucleus and large membrane‐filled cavities in the chromatin occurring in addition to craters in the equatorial region of the nucleus.
Sequences of the CyIIIa actin gene regulatory domain bound specifically by sea urchin embryo nuclear proteins
Authors: Thézé Nadine , Calzone Frank J. , Thiebaud Pierre , Hill Ronald L. , Britten Roy J. , Davidson Eric H.
Published: February 2005

Expression of the Cylla cytoskeletal actin gene is a marker of differential gene activation in the aboral ectoderm of the early sea urchin embryo. Gene transfer expreiments have defined a 2,300 nucleotide cis‐regulatory domain required for the correct spatial and temporal control of this gene. This domain includes at least 20 sites at which relatively stable DNA‐protein complexes form in vitro on reaction with embryo nuclear extracts. We report the nucleotide sequence of the whole regulatory domain and map the sites at which high‐specificity DNA‐protein interactions occur. These were located initially by gel shift assays carried out on progressive restriction digests of given subfragments of the large regulatory domain and were located more exactly by oligonucleotide gel shift competitions. Eight of the sites of specific interaction are unique within the Cyllla regulatory domain, and the remainder consist of five different sites that occur more than once. We observe some well known sequences also found in regulatory regions of other genes, e.g., “CCAAT” and “octamer” elements. The various sites have been classified regarding putative biological function in other work, and the present studies permit an assessment of the number and complexity of interactions constituting each functional class and of the relative locations of sites of each class.
Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro
Authors: Kwon Hyuk B. , Park Hyun J. , Schuetz Allen W.
Published: February 2005

Previous studies have demonstrated that direct or indirect elevation of cAMP levles in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation‐inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone‐ and frog pituitary homogenate (FPH)‐induced oocyte maturation. Similar inhibitory results were obtained when hormone‐treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6–8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation‐inducing steroid and secondarily allow its effects on the oocyte to be expressed.
Protamine amount and cross linking in mouse teratospermatozoa and aneuploid spermatozoa
Authors: De Boer P. , Redi C. A. , Garagna S. , Winking H.
Published: February 2005

The level of SH‐group oxidation in spermatozoa from the cauda epididymis was measured by a cytofluorometric method in chromosomally normal mice and two chromosome mutants. The first one, a tertiary trisomic karyotype (Ts(113)7OH), is characterized by severe oligospermia and high levels (≈75%) of malformed spermatozoa. The second, a hybrid between two European feral mouse stocks, is heterozygous for multiple Robertsonian translocations and produces exclusively aneuploid spermatozoa. Neither the severe teratospemiogenesis nor the severe aneuploidy was reflected in total SH‐group fluorescence values nor in free SH‐group fluorescence. It is concluded that both the production of protamines and protamine cross linking by S‐S bridge formation are rather autonomous processes during spermatogenesis because (1) the increased DNA variance of the aneuploid spermatozoa is not reflected in an increased variance of the total and free SH‐groups, (2) aneuploidy for the protamine gene carrying chromosome 16 is not reflected by the SH‐group values for individual spermatozoa, and (3) protamine production and cross linking are independent of the mild to severe terataspermiogenesis in the tertiary trisomic karyotype.
Temporal and spatial transcriptional regulation of the aboral ectoderm‐specific spec genes during sea urchin embryogenesis
Authors: Tomlinson Craig R. , Klein William H.
Published: February 2005

mRNAs for Spec 1 and Spec 2 of Strongylocentrotus purpuratus and LpS1 of Lytechinius pictus accumulate only in the aboral ectoderm of developing embryos. In vitro nuclear transcription assays were done to study the transcriptional regulation of these cell type‐specific genes. Spec 1, Spec 2c, and Spec 2d genes all appeared to be transcriptionally activated at the late cleavage‐early blastula stage of S. purpuratus. Differences in the relative transcription rates during development appeared to play a major role in determining the relative levels of the various Spec mRNAs. The L. pictus LpS1 gene was transcriptionally activated at a similar developmental time as the corresponding S. purpuratus genes. Nuclei from gastrula or pluteus ectodermal and endodermal/mesodermal cell fractions were used to demonstrate that Spec 1 and LpS1 genes were transcriptionally active in ectoderm nuclei but not in endoderm/mesoderm nuclei, suggesting that in vivo the Spec 1 and LpS1 genes are spatially controllod at the transcriptional level. Estimations of the absolute rate constants for Spec 1 transcription were mado at the late cleavage, mesenchyme blastula, and midgastrula stages. Calculations using these rate constants and the known levels of Spec 1 mRNA suggested that Spec 1 mRNA stability gradually increased throughout development.
Identification of extracellular proteins in the rat cumulus oophorus
Authors: Virji Nassim , Phillips David M. , Dunbar Bonnie S.
Published: February 2005

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte–cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by highresolution two‐dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postvulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW ∼81,000 and 100,000). The other two proteins were more basic and occurred at MW ∼90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.
Penetration of intact bovine ova with ram sperm in vitro
Authors: Slavík T. , Pavlok A. , Fulka J.
Published: February 2005

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal‐looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.
Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos
Authors: Kaufman M. H. , Lee K. K. H. , Speirs S.
Published: February 2005

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G‐banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable “marker” chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated form LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to dmonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellula morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.
Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton
Authors: Macrae Thomas H. , Lange Bodo M. H. , Gull Keith
Published: February 2005

The cytoskeleton exerts a direct effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecules. We have prepared six monoclonal antibodies to Triton X‐100‐insoluble components of the bull sperm cytoskeleton. One of the antibodies reacts with a detachable portion of the bull sperm acrosome. The remainder include an antibody that recognizes the principal and end piece of the tail and another that is specific to the middle piece. Two of the antibodies yield dissimilar staining patterns of the neck region and the tail, and the final monoclonal antibody stains the subacrosomal region and a detachable acrosomal domain of bull sperm. The cross reactivities of the antibodies with hamster sperm and PtK2 cells are described, as is the recognition of bull sperm polypeptides on western blots. The results suggest that these antibodies will provide interesting insights concerning the role of the cytoskeleton in sperm development and function.
Effects of gossypol on the motility of mammalian sperm
Authors: Porat Offie
Published: February 2005

The effects of the male contraceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent.
Expression of a sperm protein gene during spermatogenesis in mammalian testis: An in situ hybridization study
Authors: Wang Linfang , Miao Shiying , Yan Yuanchang , Li Yuehua , Zong Cong , Koide S. S.
Published: February 2005

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP‐B protein. Antibodies were raised against rSMP‐B protein and used to isolate and identify the cDNA coding the rSMP‐B protein from a rat testis λgt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single‐stranded 35S‐labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP‐B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP‐B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP‐B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.
Survival of bisected rabbit morulae transferred to synchronous and asynchronous recipients
Authors: Yang Xiangzhong , Foote R. H.
Published: February 2005

The rabbit was used as a model to test the concept that temporal asynchrony is required to establish physiological synchrony when embryos are bisected to produce demiembryos. In preliminary studies with intact embryos it was confirmed that embryos harvested on days 2,3,4, or 5 (day 0 = day of breeding) can be transferred with ± 1 day of asynchrony to the uteri of recipient rabbits. Three experiments were conducted with bisected embryos. In experiment 1, 192 bisected and 194 control day 3 embryos were transferred to uteri of day 2,2.5, and 3 recipients (ovulated 0, 12, and 24 h after the donors), with 14% of the bisected and 39% of the intact embryos (P < .05) resulting in young. Only 4% (2/48) of the day 3 bisected embryos vs. 39% (P < .05) of the intact day 3 embryos survived in the uteri of day 2 recipients. In experiment 2, day 3 bisected and intact embryos were transferred to the oviducts of day 3, 3.5, or 4 recipients, the speculation being that the oviduct might provide a more neutral enviroment than the uterus. However, embryo survival was very low, except for the intact embryos transferred to synchronized recipients (42% young born). In experiment 3, 150 intact and 162 (81 pairs) bisected day 3 embryos collected from uteri were transferred to uteri of day 2.5, 3.0, and 3.5 recipient does. Significantly more pregnancies (100% vs. 47%, P < .01) and young born (56% vs. 19%, P < .01) resulted from intact embryos than from bisected embryos, irrespective of the uterine age. Asynchrony (±0.5 days) did not affect pregnancy rate or young born from day 3 intact embryos, but bisected embryos are more sensitive to uterine or oviductal environment than whole embryos; survival was higher (P < .05) when they were transferred to day 2.5 recipients.