bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphibian parathyroids: Morphological and functional aspects
Authors: Srivastav Ajai Kumar , Das Vijai K. , Das Shobha , Sasayama Yuichi , Suzuki Nobuo
Published: February 2005

Amphibians living partially or totally in a terrestrial environment are the first tetrapods to possess parathyroid glands. Purely aquatic amphibians and amphibian larvae lack these endocrine glands. The parathyroids develop at the time of metamorphosis. The parathyroid glands in caecilians consist of a single cell type, that of urodeles may be composed of basal (supporting) cells and suprabasal (chief) cells, and that of anurans of small and large chief cells. Parathyroid glands of caecilians and anurans lack connective tissue, blood vessels, and nerves. The parathyroid cells become activated in response to decreased blood calcium concentration and undergo changes indicating increased parathyroid hormone secretion. Increased blood calcium concentration suppresses secretory activity. Usually, parathyroidectomy elicits hypocalcemia in most amphibians. Such operations have no effect in lower urodeles. Parathyroid hormone administration provokes hypercalcemia in most amphibians. The parathyroids of caecilians have not been studied in detail. The urodeles and anurans exhibit seasonal changes in the parathyroid glands. These changes may be initiated by environmental stimuli such as light, temperature, or alterations in blood calcium levels caused by natural hibernation. © 1995 Wiley‐Liss, Inc.
Microvascularization of corpuscles of stannius in teleost fishes
Authors: Lametschwandtner Alois
Published: February 2005

Blood supply and microvascular patterns of Stannius corpuscles were studied by scanning electron microscopy of vascular corrosion casts in the teleost fishes Blennius pavo, Zosterisessor ophiocephalus, and Gasterosteus aculeatus. Microvascular casts demonstrated that Stannius corpuscles—depending on their location—have an arterial supply derived either directly from the dorsal aorta, from the trunk of the first ventral segmental artery of the tail, or from a renal artery. Supplying arteries form a capsular capillary bed and a parenchymal capillary bed; both are composed of fine, freely anastomosing vessels with a homogeneous isotropic distribution. Central venules arise deep in the corpuscles. In the capsule, they form a single vein which drains into a segmental vein or directly into the caudal vein. Stanniocalcin, the hormone of the Stannius corpuscle, enters the renal circulation and reaches its main target organs, the gills, via posterior cardinal veins—heart—ventral aorta. Occasionally, some capsular venules empty into the trunk kidney peritubular venules. Capillaries are fenestrated and are embraced by pericytes with long, slender processes. The perivascular space contains collagen fibrils. Nerve fibers are found close to endothelial cells and pericytes. Vascular patterns of Stannius corpuscles are compared with those of the rat parathyroid glands and are discussed in respect to physiological implications. © 1995 Wiley‐Liss, Inc.
Ultrastructure of human parathyroid cells in health and disease
Authors: Cinti Saverio , Sbarbati Andrea
Published: February 2005

Parathyroid glands (n = 271) removed from 130 patients were examined by light and electron microscopy. A standardized method of tissue processing was employed and morphometry was performed. The aim of the paper is to provide a description of the human parathyroid chief cell ultrastructure in health and disease, with quantitative evaluation of structures involved in secretion of parathyroid hormone in a large case series, and to discuss their role in current diagnostic histopathology. The patients were euparathyroid (n = 10), or affected by primary (n = 97), secondary (n = 8), or tertiary (n = 15) hyperparathyroidism. In normal glands, solid parenchyma was composed of chief cells, large clear cells, transitional‐oxyphil cells, and oxyphil cells. Chief cell hyperplasia, pseudo‐adenomatous hyperplasia, adenoma, water‐clear cell hyperplasia, and carcinoma were the most usual forms of parathyroid disease responsible for primary hyperparathyroidism. In chief cell hyperplasia, all the parathyroid glands were enlarged and the chief cells were in an active state of hormone secretion, with a large Golgi complex, abundant rough endoplasmic reticulum (RER), small lipid droplets, and tortuous plasma membrane. In pseudo‐adenomatous hyperplasia, one gland was enlarged and the others displayed a normal size; however, electron microscopic examination and morphometric analysis showed that all the glands had active cells. Adenomas displayed a pattern similar to those of pseudo‐adenomatous hyperplasia, with one gland enlarged and the others of normal size. However, ultrastructural examination and morphometry showed that the normal‐size glands were hypo‐active. Water‐clear cell hyperplasia showed cells filled with cytoplasmic vacuoles. In these cells, structures with intermediate features between secretory granules and vacuoles were visible. Nucleo‐cytoplasmic atypias were frequently visible in parathyroid carcinoma cells. In secondary and tertiary hyperplasia, active chief cells were regularly mixed with oxyphil or transitional‐oxyphil cells. The tertiary hyperplasia was characterized by RER‐associated structures that were not found in the normal or other pathologic conditions. These results demonstrate that electron microscopy and morphometry represent useful tools in parathyroid histopathology. © 1995 Wiley‐Liss, Inc.
Spontaneous and induced remyelination in multiple sclerosis and the theiler's virus model of central nervous system demyelination
Authors: Miller David J. , Rodriguez Moses
Published: February 2005

Remyelination in the central nervous system, originally thought to occur rarely, if ever, is now an established phenomena in multiple sclerosis patients. However, the extent of myelin repair is incomplete and limited. Experimental models of central nervous system demyelination provide an opportunity to study the cellular and molecular events involved in remyelination. These models may provide some clue to why remyelination in multiple sclerosis is incomplete as well as suggest potential methods to stimulate central nervous system repair. In this review we examine the morphological aspects of central nervous system remyelination and discuss both spontaneous and induced remyelination in multiple sclerosis and experimental models of central nervous system demyelination. We give special emphasis to the Theiler's virus model of central nervous system demyelination and its usefulness to identify therapeutic agents to promote remyelination. The role of immunoglobulins in promoting remyelination in both the Theiler's model system and in multiple sclerosis is discussed. Finally, we examine the potential physiological role of demyelination and remyelination and its relationship with clinical manifestations of central nervous system disease. © 1995 Wiley‐Liss, Inc.
Microwave fixation: Understanding the variables to achieve rapid reproducible results
Authors: Giberson Richard T. , Demaree Richard S.
Published: February 2005

The use of microwave irradiation for rapid chemical fixation of tissues in electron microscopy is a subject of current interest. The effect of water load size and location, sample placement in the oven cavity (hot or cold spots), and time on tissue preservation were examined. The use of a microwave container (4 dram vial) encased in 60 ml of ice in a 100 ml polyethylene beaker and a 0% power setting between two 100% power settings (time interval) provided reliable control of temperature during microwave irradiation. High brightness neon lights provided a quick and easy method to identify and map hot and cold spots within the oven cavity. Using microwave irradiation for rapid glutaraldehyde and osmium tetroxide fixation of tissues (Pacific yew needle and mouse kidney and liver) for electron microscopy yielded preservation equal or better than routine immersion fixation when a time interval, a cold spot (as the sample location), and an ice‐encased vial were used during microwave fixation. These adaptations provided reliable control of fixation conditions in an 800 watt laboratory microwave oven. © 1995 Wiley‐Liss, Inc.
Interpreting MHC class I expression and class I/class II reciprocity in the CNS: Reconciling divergent findings
Authors: Lampson Lois A.
Published: February 2005

MHC‐restricted T cells are thought to contribute to clinical demyelination in MS and other circumstances. The step‐by‐step mechanisms involved and ways of controlling them are still being defined. Identification of the MHC+ cells in the CNS in situ has been controversial. This chapter reviews MHC expression in neural tissue, including normal, pathological, experimental, and developing tissue in situ and isolated cells in vitro. A basic pattern is defined, in which MHC expression is limited to nonneural cells and strongest class I and II expression are on different cell types. Variations from the basic pattern are reviewed. Ways of reconciling divergent findings are discussed, including the use of “mock tissue” to help choose between technical and biological bases for divergent findings, the potential contribution of internal antigen to the in situ staining patterns, and the possibility that class I upregulation is actively suppressed in situ. Functional implications of the observed patterns of MHC expression and ways of confirming the function of each MHC+ cell type in situ are described. It is suggested that modulating MHC expression in different cell types at different times or in different directions might be desirable. © 1995 Wiley‐Liss, Inc.
Progressive multifocal leukoencephalopathy: A review of the pathology and pathogenesis
Authors: Aksamit Allen J.
Published: February 2005

Progressive multifocal leukoencephalopathy is an important viral opportunistic infection of oligodendrocytes leading to direct demyelination. Virus is likely disseminated to the brain via the blood. However, the timing of that dissemination with relationship to clinical disease is unknown. Important clues about viral pathogenesis have been learned by applying molecular in situ techniques to diseased brain. The oligodendrocyte is the primary target for JC virus infection, and its death is the primary reason for demyelination. Bizarre astrocytes show limited viral DNA replication but are abortively infected. Although lymphoid organs can be infected by JC virus, there is no definitive evidence that lymphoid cells carry virus into the brain at the time of immunosuppression. JC virus may be reactivated from a latent state in both the brain and in non‐central nervous system (CNS) organs at the time of immunosuppression, leading to clinical disease. Future sensitive in situ studies will likely resolve controversies about pathogenesis. © 1995 Wiley‐Liss, Inc.
Freeze‐Fracture, Deep‐Etch, and Freeze‐Substitution Studies of Olfactory Epithelia, With Special Emphasis on Immunocytochemical Variables
Authors: Menco Bert
Published: February 2005

Freeze‐fracturing and deep‐etching are a well‐suited set of methods to study membrane and cytoplasmic features. Various approaches are available. Possible variables include tissue preparation, fracturing only or fracturing followed by etching, modes and materials of replication, and various ways of combining freeze‐fracturing and/or deep‐etching with (immuno)cytochemistry. Freeze‐substitution, in particular combined with embedding in methacrylate resins such as the Lowicryls, is becoming rather widely accepted for purposes of ultrastructural (immuno)cytochemistry. Most investigators active in this field agree that this combination yields superior results compared to (immuno)cytochemistry combined with more conventional means of thin section transmission electron microscopy. Yet relatively little information is available on the variations that can occur with different approaches of freeze‐substitution immunocytochemistry. This review deals with some of the variations in freeze‐fracturing, freeze‐etching, and freeze‐substitution as applied to olfactory epithelial structures and with the effectiveness of observations obtained by application of the above sets of methods in relating the special morphology of olfactory epithelial cellular structures with those obtained by other approaches. Indeed, the data obtained continue to provide an integral image in which that morphology can be related to the special biochemistry, cell and molecular biology, and electrophysiology of olfactory epithelial structures. © 1995 Wiley‐Liss, Inc.
Colocalization of mRNA and protein using in situ hybridization and immunohistochemistry in testicular tissue
Authors: Millar M. R. , Sharpe R. M. , Maguire S. M. , Gaughan J. , Saunders P. T. K.
Published: February 2005

Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein‐1 (TP‐1) and sulfated glycoprotein‐1 (SGP‐1), respectively. It was found that SGP‐1 could be detected in tissue sections subsequent to the detection of TP‐1 mRNA in situ. The finding that (1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and (2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized. © 1995 Wiley‐Liss, Inc.
Novel Biomimetic Surfactant: Synthesis and Micellar Characteristics
Authors: Xu Jian‐Ping , Ji Jian , Chen Wei‐Dong , Shen Jia‐Cong
Published: February 2005

Summary: Novel biomimetic surfactants based on cholesterol as the hydrophobic segment and poly[2‐(methacryloyloxy)ethyl phosphorylcholine] (pMPC) as the hydrophilic segment were synthesized in the present study by atom transfer radical polymerization (ATRP) of 2‐(methacryloyloxy)ethyl phosphorylcholine (MPC) using a cholesterol‐based macroinitiator. The association behavior of cholesterol‐block‐poly[2‐(methacryloyloxy)ethyl phosphorylcholine] (Chol‐pMPCs) in aqueous solution was studied by 1H NMR spectroscopy, fluorescence probe technique, and atomic force microscopy (AFM). The 1H NMR spectrum of the polymer in CD3OD showed both the cholesterol group and the phosphorylcholine group while the cholesterol group did not appeared in the 1H NMR spectrum of the polymer in D2O, which implied the formation of a micelle structure. Fluorescence excitation spectra of a pyrene probe solubilized in the aggregates of Chol‐pMPCs suggested the presence of a critical micelle concentration (cmc) in water. The critical micelle concentrations of the polymers CMPC10, CMPC20 and CMPC40 were determined to be 7.27 × 10−3, 13.47 × 10−3, and 20.77 × 10−3 mg · mL−1, respectively. AFM images of the aggregates on mica suggested that the pMPC block formed the biocompatible micelle coronas and the cholesterol block formed the hydrophobic micelle cores. These new biomimetic diblock copolymers were evaluated as “stealthy” nanocapsules for the delivery of hydrophobic drugs. The anti‐cancer drug adriamycin (ADR) was chosen as a hydrophobic drug to be incorporated into the inner core of the micelles and the morphology of the drug‐loaded micelles were observed by AFM.

Schematic of the biomimetic block copolymers and their micellization, and an AFM image of the micelles deposited on mica.

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Schematic of the biomimetic block copolymers and their micellization, and an AFM image of the micelles deposited on mica.
Mechanism of Conjugated Polymer Organization on SWNT Surfaces
Authors: Narizzano Riccardo , Nicolini Claudio
Published: February 2005

Summary: The interactions between poly{(2,6‐pyridinylenevinylene)‐co‐[(2,5‐dioctyloxy‐p‐phenylene)vinylene]} (PPyPV) and SWNTs have been investigated using UV‐Vis absorption spectroscopy. The SWNTs promoted polymer organization. PPyPV is a Lewis base and can be doped by strong and weak Lewis acids. The basicity strength of the PPyPV depended on the polymer interchain interactions, which were enhanced by the presence of SWNTs. As the SWNT concentration was increased, an increment in the Kb of PPyPV was observed.

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Re‐Gradation of Photo‐Oxidized Post‐Consumer Greenhouse Films
Authors: La Mantia Franceso P. , Dintcheva Nadka Tzankova
Published: February 2005

Summary: The degradation undergone by the polymers during their use and because of the thermo‐mechanical degradation undergone during the reprocessing operations provokes, among other effects, the decrease of the molecular weight. The change of the molecular architecture is responsible for the deterioration of all the properties. In order to restore the properties of post‐consumer recycled plastics, some rebuilding of the molecular structure is necessary. In this work, photo‐oxidized films for greenhouses were reprocessed in presence of additives able to react with the polyethylene in order to form branching and cross‐linking, improving the properties of these post‐consumer plastic.

Pressure as a function of time in the mini twin‐screw extruder, processing speed 70 rpm.

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Pressure as a function of time in the mini twin‐screw extruder, processing speed 70 rpm.
Low‐Band Gap Polyplatinaynes with Electron‐Accepting Silole Units
Authors: Wong Wai‐Yeung , Wong Chun‐Kin , Poon Suk‐Yue , Lee Albert W.‐M. , Mo Tian , Wei Xin
Published: February 2005

Summary: The synthesis and thermal, redox and photoluminescence properties of a soluble donor‐acceptor polyplatinayne with the electron‐accepting silole ring and its model compound are described. The polymer has an optical band gap of 2.10 eV which is much lower than that of thienyl‐ or silyl‐bridged congeners. The incorporation of electron‐accepting silole unit in the metallopolymer main chain creates a new π‐conjugated system that features unique donor‐acceptor characteristics.

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Clinical and genetic evaluation in a French population presenting with primary focal dystonia
Authors: Dhaenens Claire‐Marie , Krystkowiak Pierre , Douay Xavier , Charpentier Pierre , Bele Sylvain , Destée Alain , Sablonnière Bernard
Published: February 2005

Primary focal dystonia (PFD) is known to be a clinically and genetically heterogeneous group of movement disorders. To evaluate the frequency of familial focal dystonia in a French population presenting with PFD, we screened 197 patients (150 index cases and 47 affected family members) presenting focal primary dystonia for the GAG deletion in the DYT1 gene and analyzed linkage to the DYT6, DYT7, and DYT13 loci in those who presented a family history. Fourteen families could be recruited and, among them 47 new symptomatic individuals could be identified by clinical examination. A group of 104 patients were without family history and 46 patients (30.7%) were found to have at least one first‐degree relative with dystonia. Mean age at onset was significantly later (55.4 ± 14.0 years) in the blepharospasm group and earlier in patients with writer's cramp (35.8 ± 14.0 years). The group of patients with family history showed a mean age at onset significantly earlier (39.2 ± 18.0) than in patients without family history (47.4 ± 14.4 years). Fourteen families demonstrated an autosomal mode of transmission and five families were studied further for genetic linkage analysis, but no significant linkage to one of the three loci could be observed. Our results illustrate the importance of genetic factors and the clinical heterogeneity of PFD. They indicate the existence of one or several as yet unmapped genes responsible for these diseases. © 2005 Movement Disorder Society
Dopaminergic innervation of the human striatum in Parkinson's disease
Authors: Porritt Michelle , Stanic Davor , Finkelstein David , Batchelor Peter , Lockhart Sharon , Hughes Andrew , Kalnins Renate , Howells David
Published: February 2005

In Parkinson's disease (PD), dopaminergic input to the caudate nucleus and a band of putaminal tissue abutting the external globus pallidus seems well preserved on immunohistochemical staining for the dopamine transporter. Counting of dopaminergic terminals showed that terminal density in these regions in PD was the same as that in controls, which indicates that input is truly preserved and not a consequence of a compensatory upregulation of metabolism in a reduced pool of surviving terminals. When the branching pattern of dopaminergic axons coursing through the globus pallidus was examined, we found no evidence for increased axonal sprouting in PD that might have contributed to preservation of dopaminergic input to the putamen or caudate nucleus. Although terminal counting indicated that anatomic input was preserved to parts of the striatum, dopamine uptake site density in these regions was reduced significantly. This suggests that the impact of disease in these areas is more profound than was thought previously. © 2005 Movement Disorder Society
Parkinsonism in the course of HTLV‐I‐associated myelopathy
Authors: Puccioni‐Sohler Marzia , Papais‐Alvarenga Regina , de Souza Priscila Mesquita , de França Silvia Curi , Gonçalves Reizer Reis , Jacobson Steven
Published: February 2005

Parkinsonian syndromes may represent a complication of viral infection. Human T cell lymphotropic virus I (HTLV‐I) is a cause of a chronic myelopathy in which encephalic involvement has been also found. We report on the case of a 60‐year‐old man with HTLV‐I‐associated myelopathy, complicated with bradykinesia, resting tremor, and cogwheel rigidity. These findings suggest that parkinsonian features may represent a neurological disorder associated with HTLV‐I infection. © 2005 Movement Disorder Society
Mucin‐like glycoproteins in the equine embryonic capsule
Authors: Oriol Julio G. , Betteridge K. J. , Clarke A. J. , Sharom F. J.
Published: February 2005

The equine embryonic capsule replaces the zona pellucida and envelops the conceptus during the second and third weeks of pregnancy. Although this capsule was described more than 100 years ago, its molecular structure has not been characterized. Here we present evidence that the glycoprotein(s) of the equine capsule resembles those of the mucin glycoprotein family. The resistance of the capsule to chemical and enzymatic solubilization was confirmed, and, as in mucins, protein constituted only 35–40% of its total dry mass. Determination of the sugar composition of the capsule using colorimetric assays and high‐performance anion‐exchange chromatography also showed it to have mucin‐like characteristics. Gal, GalNAc, sulfated sugars, and sialic acid make up a high proportion of the capsular carbohydrate, while GlcNAc, Glc, and Man are minor components. These findings were verified using lectin histochemical staining of frozen sections of conceptuses. The results of amino acid analysis were also consistent with the proposal that the capsular glycoproteins belong to the mucin family. Removal of the covalently bound carbohydrate by b̃‐elimination under reducing conditions demonstrated that the capsule is O‐glycosylated mainly on threonine residues. Affinity chromatography on jacalin‐agarose confirmed that, like mucins, the capsular glycoproteins are heavily O‐glycosylated. SDS‐PAGE analysis revealed a prominent 21‐kDa band, specific to the capsule, in preparations solubilized by trypsin but not by other proteases. Characterization of its constituent glycoprotein(s) should be helpful in elucidating the role of the capsule (and analogous blastocyst coverings in other species) during early pregnancy. © 1993 Wiley‐Liss, Inc.
The cleavage rate of digynic triploid mouse embryos during the preimplantation period
Authors: Henery C. C. , Kaufman M. H.
Published: February 2005

Triploidy is a lethal condition in mammals, with most dying at some stage between implantation and term. In humans, however, a very small proportion of triploids are liveborn but display a wide range of congenital abnormalities. In particular, the placentas of human diandric triploid embryos consistently display “partial” hydatidiform molar degeneration, while those of digynic triploids generally do not show these histopathological features. In mice, the postimplantation development of diandric and digynic triploid embryos also differs. While both classes are capable of developing to the forelimb bud stage, no specific degenerative features of their placentas have been reported. Diandric triploid mouse embryos are morphologically normal while digynic triploid mouse embryos consistently display neural tube and occasionally cardiac abnormalities.

Previously it was shown that the preimplantation development of micromanipulated diandric triploid mouse embryos was similar to developmentally matched diploid control embryos. In this study, the preimplantation development of micromanipulated digynic triploid mouse embryos is analysed and compared with that of diandric triploid mouse embryos in order to determine whether there is any difference in cleavage rate between these two classes of triploids.

Standard micromanipulatory procedures were used to insert a female or a male pronucleus into a recipient diploid 1‐cell stage embryo. The karyoplast was fused to the cytoplasm of the embryo by electrofusion. These tripronucleate 1‐cell stage embryos were then transferred to pseudopregnant recipients and, at specific times after the HCG injection to induce ovulation, the embryos were recovered and total cell counts made. These results were plotted and regression lines drawn.

An additional control group of embryos was subjected to similar micromanipulatory procedures to those used in the experimental study. These embryos had a single pronucleus removed and this was then reinserted into the perivitelline space. Diploidy was immediately restored by electrofusion. These embryos were transferred to recipients and at specific times after the HCG injection the embryos were recovered and total cell counts made. These results were also plotted and regression lines drawn.

The results show that the cell doubling time of the digynic triploid embryos was 14.84 h (± 1.19). This was not significantly different from that of the diandric triploid embryos (13.55 h ± 0.86; P > 0.05) or of the manipulated diploid controls (12.12 h ± 0.79; P > 0.05). © 1993 Wiley‐Liss, Inc.
Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology
Authors: Huszar Gabor , Vigue Lynne
Published: February 2005

Our previous creatine phosphokinase (CK) activity studies in human sperm revealed differences among men and among sperm populations within the same specimen. Samples with low sperm concentrations, high incidence of abnormal sperm morphology, and diminished fertility had higher per sperm CK activity. In the present work, we demonstrated, with 14C‐FDNB covalent CK active site modification and with direct CK immunocytochemistry, that the higher CK activity is related to an increased content of CK and of other proteins in sperm. Also, sperm heads with higher CK content were significantly larger and rounder and showed a higher incidence of amorph configuration. We suggest that these biochemical and morphological irregularities are related and are due to a failure of spermatogenesis, more specifically, to a higher retention of cytoplasm, which in normal sperm development is lost to the Sertoli cells as residual bodies. Thus higher CK activity and larger or irregular head size in human sperm signify cellular immaturity and a failure to complete spermatogenesis. © 1993 Wiley‐Liss, Inc.
Simultaneous detection of X‐ and Y‐bearing human sperm by double fluorescence in situ hybridization
Authors: Han Tie Lan , Ford Judith H. , Webb Graham C. , Flaherty Sean P. , Correll Anthony , Matthews Colin D.
Published: February 2005

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin‐labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immuno‐cytochemically, using two different and independent affinity systems. Ninety‐six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX‐bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY‐bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X‐ and Y‐bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY‐bearing sperm, 0.62% were diploid XY‐bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X‐chro‐mosome and Y‐chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y‐bodies. © 1993 Wiley‐Liss, Inc.
Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight‐cell embryos matured and fertilized in vitro
Authors: Mitani Tasuku , Utsumi Kyozo , Iritani Akira
Published: February 2005

Single blastomeres from eight‐cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two‐ to eight‐cell stage 48 hr after electrofusion was increased when both the eight‐cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol‐treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two‐ to eight‐cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley‐Liss, Inc.
Transgenic strategies in reproductive endocrinology
Authors: Kelley Kevin M. , Johnson Thomas R. , Gwatkin Ralph B. L. , Ilan Judith , Ilan Joseph
Published: February 2005

The present discussion surveys some of the recently published studies utilizing transgenic strategies to address questions in reproductive endocrinology. Beginning with a brief introduction of the transgenic method itself, the following areas are covered: 1. Sexual development and Müllerian‐inhibiting substance; 2. Hypogonadal mice and hypothalamic GnRH; 3. The GnRH neuron: generation of immortalized rare cell types; 4. Glyco‐protein hormones: immortalized cells, development and evolution; 5. Growth hormone and reproduction; and, 6. Gestation and the insulin‐like growth factors. In each section, the discussion attempts to be integrative with respect to the significance of the results to physiological, cellular and molecular biology. We believe this approach is appropriate, as transgenic science itself is necessarily an integration of all of these levels of investigation and participation from those working at all levels is needed. © 1993 Wiley‐Liss, Inc.
Progressive expression of trophoblast‐specific genes during formation of mouse trophoblast giant cells in vitro
Authors: Carney E. W. , Prideaux V. , Lye S. J. , Rossant J.
Published: February 2005

The expression of a battery of trophoblast‐specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin‐related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL‐I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley‐Liss, Inc.
Process of nuclear envelope reduction in spermiogenesis of a mosquito, Culex tigripes
Authors: Ndiaye Mady , Mattei Xavier
Published: February 2005

When the Culex tigripes spermatid begins to elongate, the nucleus exhibits on its surface invaginations of the nuclear envelope. These invaginations have a uniform diameter of 0.3 μm. They separate from the envelope of the nucleus and form spherical intranuclear vesicles. In the old spermatids these vesicles are imprisoned in the condensed chromatin. The spermatozoon also possesses these vesicles which are then ovoid in shape. This process of vesiculation permits the diminution of the surface of the nucleus when it decreases in volume during spermiogenesis. © 1993 Wiley‐Liss, Inc.
Control of in vitro maturation of bovine cumulus‐oocyte complex by preovulatory granulosa cells
Authors: Rabahi Flora , Monniaux Danielle , Pisselet Claudine , Durand Philippe
Published: February 2005

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus‐oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100‐fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five‐ and 500‐fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley‐Liss, Inc.
Phosphorylcholine‐binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma
Authors: Leblond E. , Desnoyers L. , Manjunath P.
Published: February 2005

The major proteins of bovine seminal plasma, BSP‐A1, BSP‐A2, BSP‐A3, and BSP‐30kDa (collectively named BSP proteins) bind to phospholipids containing the phosphorylcholine moiety. An affinity purification method using a p‐aminophenyl phosphorylcholine‐Agarose (PPC‐Agarose) affinity matrix was developed for their purification. In this study, we investigated the distribution of BSP‐like analogues in seminal fluid of the human, porcine, hamster, mouse, and rat using this affinity matrix. Alcohol precipitates of the seminal plasma/seminal vesicle secretions (SP/SVS) were further delipidated using isopropyl ether:n‐butanol (60:40). The protein preparations obtained were solubilized in a minimal volume of buffer A (50 mM Tris‐HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% NaN3), dialyzed against the same buffer, and applied to a PPC‐Agarose column connected to a FPLC system. The unbound material was washed out and the adsorbed proteins eluted with buffer A containing 10 mM phosphorylcholine (PrC) and 10 M urea. The fractions were separated by SDS‐PAGE, stained or transferred onto a nitrocellulose membrane, and probed with rabbit polyclonal anti‐BSP antibodies. Anti‐BSP cross‐reacting proteins were detected in the seminal fluids of all the species investigated. Moreover, many of these proteins bound to the affinity matrix. The BSP proteins and their immunoreacting analogues appear to be ubiquitous in mammals and may possibly be involved in a common function such as in the modification of the lipid content of the sperm plasma membrane. © 1993 Wiley‐Liss, Inc.
Effects of polyamine biosynthesis inhibitors on the progesterone‐initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm
Authors: Meizel Stanley , Turner K. O.
Published: February 2005

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca2+([Ca2+]i) and an increase in a Ca2+‐requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca2+ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone‐initiated increase in [Ca2+]i and AR in capacitated human sperm in vitro: DL‐α‐(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′‐{[(Z))‐4‐amino‐2‐butenyl]methylamino}‐5′‐deoxyadenosine (MDL 73811), an inhibitor of S‐adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein‐labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone‐initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone‐initiated increase in [Ca2+]i (assayed with fura‐2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca2+]i nor did preincubation with either polyamine followed by progesterone addition increase [Ca2+]i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone‐initiated rapid increase in [Ca2+]i and subsequent membrane events of the AR. © 1993 Wiley‐Liss, Inc.
PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro
Authors: Valdivia Ruby P. A. , Kunieda Tetsuo , Azuma Sadahiro , Toyoda Yutaka
Published: February 2005

A two‐step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast‐intermediate‐ and slow‐growing embryos, a significantly high percentage (78.0%) of the fast‐developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow‐developing embryos were identified as males. The intermediate‐developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast‐ and slow‐developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos. © 1993 Wiley‐Liss, Inc.
High frequency of DNA ploidy abnormalities in preimplantation embryos of the rabbit
Authors: Schumacher Armin , Fischer Bernd , Agorastos Theodoros , Beier Henning M.
Published: February 2005

The DNA ploidy of Feulgen‐stained cell nuclei of in vivo preimplantation rabbit embryos was assayed by cytophotometry. DNA ploidy abnormalities were detected in single‐cell nuclei readings by the criterion of ≥5C DNA. These hypermodal DNA contents are referred as to DNA aneuploidy. Two, 4 and 6 days old rabbit embryos, all of normal gross morphology, were investigated.

The incidence of embryos with DNA ploidy abnormalities increased from 17% in 2‐day‐old cleavage stages to 51% in 6‐day‐old expanded blastocysts. All these embryos were mosaics and the percentage of DNA aneuploid nuclei per embryo did not usually exceed 9%. Fifteen percent of the expanded blastocysts, however, contained up to 23% abnormal nuclei. Throughout the embryonic stages studied, the DNA content of abnormal nuclei was remarkably constant and averaged 5.8C. DNA aneuploid and euploid blastocysts did not differ in size. A maternal FSH treatment did not influence the DNA ploidy.

This is the first report on the DNA ploidy pattern in preimplantation rabbit embryos. Our results indicate that DNA aneuploidy of single blastomeres is common in this species and occurs more often than generally assumed. The embryonic viability does not seem to be affected by the presence of DNA aneuploid blastomeres supporting earlier findings that a limited number of abnormal blastomeres is compatible with normal preimplantation development. © 1993 Wiley‐Liss, Inc.
Sites of transcription of the Müllerian inhibiting substance gene in mouse testis
Authors: Ao Asangla , Erickson Robert P. , Stalvey John R. D.
Published: February 2005

We have examined the transcription of Müllerian inhibiting substance (MIS) in testis by the sensitive technique of reverse transcription polymerase chain reaction (RTPCR). A developmental study of testis by this nonquantitative technique showed expression at all postnatal stages, including adults while liver and kidney provided negative controls. Cell separation studies indicated that highly purified interstitial cells, as well as less homogeneous Sertoli cell‐enriched and germ cell‐enriched fractions, contained RNA for MIS. The transcription of MIS in an interstitial cell type was confirmed by finding MIS mRNA in purified Leydig cells. Inasmuch as the germ cell‐enriched fraction contains some Sertoli cells, and XX,Sxra and XX,Sxrb which have germ cell‐depleted testes, contain MIS mRNA, a Sertoli cell source remains likely for the seminiferous tubule compartment. © 1993 Wiley‐Liss, Inc.
Cholesterol and polyunsaturated acid enriched diet: Effect on kinetics of the acrosome reaction in rabbit spermatozoa
Authors: Diaz‐Fontdevila Marina , Bustos‐Obregon Eduardo
Published: February 2005

In this study we examined the effect of cholesterol (Diet 2), cholesterol and fish oil (FO) polyunsaturated acid (Diet 3), and polyunsaturated acid (Diet 4) enriched diets upon the acrosome reaction (AR) of New Zealand White rabbit spermatozoa. Male rabbits fed with cholesterol alone or with FO increased their cholesterol and LDL‐cholesterol serum levels after 15 days of diet. Ten semen samples were obtained after 2 months of diet. Our results suggest that hypercholesterolemia and hypertriglyceridemia in male rabbits could produce a decreased capacity of sperm AR after 4 h (0%, 0%, and 60% lower than the control), 6 h (0%, 68%, and 44%), or 8 h (58%, 52%, and 32%) of incubation in capacitating medium. Another set of experiments were made with lysophosphatidylcholine (LPC), 80 μg/ml, and the same pattern of AR was seen. Nevertheless, the high cholesterol and total lipids (TL) levels in serum did not affect the cholesterol levels in seminal plasma (SP) but affect the SP total lipids. The diminished capacity of rabbit sperm to undergo the AR was not reverted by in vitro incubation with the Shinitsky medium for cholesterol depletion (MDC). These results indirectly suggest that the cholesterol/phospholipid ratio in hypercholesterolemic sperm is similar to that of controls and are in agreement with preliminary studies made in our laboratory that evidenced the same cholesterol/ phospholipid ratio in rabbit sperm from hypercholesterolemic animals than from controls. These findings and the higher sterol‐filipin complex in the acrosomal membrane of hypercholesterolemic animals compared to controls reported by Diaz‐Fontdevila et al. (1992) could suggest that in this study different sperm membrane lipid domains induced by the hypercholesterolemia are the cause of the AR kinetics modification. © 1993 Wiley‐Liss, Inc.
Binding of anti‐H‐Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm
Authors: Hendriksen Peter J. M. , Tieman Marion , Van Der Lende Tette , Johnson Lawrence A.
Published: February 2005

Studies designed to answer the question whether or not H‐Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H‐Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti‐H‐Y antibody binding to fractions enriched for X and Y chromosme bearing sperm, directly. A total of 7 anti‐H‐Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H‐Y antigen is preferentially expressed in Y chromosome bearing sperm. © 1993 Wiley‐Liss, Inc.