bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructure of the sperm and spermatogenesis and spermiogenesis of Dina lineata (hirudinea, erpobdellidae)
Authors: Bonet S. , Molinas M.
Published: February 2005

The mature sperm of Dina lineata is of the modified type. The sperm are 48 μm long and 0.3 μm wide. The sperm are filiform and helicoidal cells with a distinct head, a midpiece, and a tail. There are two distinct regions in the head: the acrosome and the posterior acrosome, each with its own characteristic morphology. The midpiece is the mitochondrial region and has a single mitochondrion. Two distinct portions can be observed in the tail: the axonematic region and the terminal piece.

In the process of spermatogenesis the early spermatogonia divide to form a poliplast of 512 spermatic cells. In the spermiogenesis the following sequential stages can be distinguished: elongation of the flagellum; reciprocal migration of mitochondria and Golgi complex; condensation of chromatin and formation of the posterior acrosome; spiralization of nuclear and mitochondrial regions; and, finally, formation of the anterior acrosome.

The extreme morphological complexity of the Dina spermatozoon is related to the peculiar hypodermal fertilization which characterizes the erpobdellid family. Correlation between sperm morphology and fertilization biology in the Annelida is revised.
Fine structure of early human embryos frozen with 1,2 propanediol
Authors: Ng S. C. , Sathananthan A. H. , Wong P. C. , Ratnam S. S. , Ho J. , Mok H. , Lee M. N.
Published: February 2005

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two‐to eight‐cell human embryos can survive slow freeze‐thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase‐contrast microscopy. We assessed the structure of 25 frozen‐thawed one‐ to 12‐cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase‐contrast and electron microscopy, using the same method of cryopreservation. One‐fourth of the embryos examined had all cells intact, and more than one‐half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell‐to‐cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.
Nuclear protein transitions in cuttle‐fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage
Authors: Rousseaux‐Prévost Roselyne , Engelhardt René‐Pierre , Rousseaux Jean , Wouters‐Tyrou Danièle , Sautière Pierre
Published: February 2005

The changes in basic nuclear proteins throughout cuttle‐fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti‐protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti‐protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle‐fish, nuclear protein transitions involve the replacement of histones by a spermatid‐specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle‐fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.
Evidence for two forms of phospholipase A2 in human semen
Authors: Antaki P. , Langlais J. , Ross P. , Guérette P. , Roberts K. D.
Published: February 2005

The molecular weight of the active unit of phospholipase A2 (PA2) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA2 activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA2 activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA2 was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA2 activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at −20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long‐term storage of spermatozoa at −20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or −20 °C) or long‐term storage and subsequent heat treatment. Thus, the behavior of PA2 when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA2 was found and was sensitive to changes in temperature.
Does phospholipase C inhibit fusion between hamster sperm and zona‐free eggs?
Authors: Clark Judy M. , Koehler James K.
Published: February 2005

Previous studies (Hirao and Yanagimachi: Gamete Res. 1:3–12, 1978) have found that phospholipase C (PLC) preparations inhibit sperm‐egg fusion. We have attempted to duplicate these results with PLC, as well as with a more specific enzyme, phosphatidyl‐inositol‐specific PLC. PLC preparations were applied externally to zona‐free hamster eggs prior to incubation with sperm. Phosphatidylinositol‐specific PLC did not inhibit sperm penetration. The degree of sperm‐egg fusion observed after egg exposure to PLC, however, was dependent upon the purity of the commercial preparation. An impure sample of PLC inhibited sperm penetration, while a more purified preparation did not. The morphology of eggs was unaffected by exposure to phosphatidylinositol‐specific PLC and the more purified PLC preparation. The impure preparation, however, was disruptive primarily to the egg plasma membrane as well as to internal organelle organization. The degree of damage by the impure PLC preparation was concentration dependent. The results suggest that as purity of the PLC preparation is increased, the adverse effects of PLC on sperm‐egg fusion become negligible.
Ultrastructural mapping of a sperm plasma membrane autoantigen before and after the acrosome reaction
Authors: Esaguy Nair , Welch Jeffrey E. , O'Rand Michael G.
Published: February 2005

The rabbit sperm plasma membrane autoantigen, RSA, is a zona binding protein that binds the spermatozoon to the zona pellucida both before and after the acrosome reaction. In the present study rabbit spermatozoa undergoing the acrosome reaction in vitro are described and monospecific polyclonal mouse anti‐RSA and protein A‐gold label is used with the label‐fracture technique (Pinto de Silva and Kan, J Cell Biol, 99:1156–1161, 1984) to map the location of RSA at the ultrastructural level before and after the acrosome reaction. RSA is most concentrated in the plasma membrane over the postacrosomal‐equatorial region border. The label appears to cluster over the anterior aspects of the postacrosomal region's tooth‐like projections. Following the acrosome reaction, RSA is still present in the postacrosomal region and often appears clustered in the medial aspects of the equatorial region.
Duration of epididymal sperm transit in hamster: An autoradiographic study
Authors: Hikim Amiya P. Sinha , Hoffer Anita P.
Published: February 2005

The time required for passage of spermatozoa through the epididymis has been determined in hamster employing quantitative light microscopic autoradiography with 3H‐thymidine. The time required for spermatozoa to traverse the caput is 3 days; corpus, 2 days; proximal cauda, 2 days; distal cauda, 6 days. An additional 2 days are required for passage of spermatozoa through the proximal ductus deferens. The total duration of sperm transit through the ductus epididymis in sexually rested hamster has been estimated at about 15.0 days.
Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation
Authors: Langlais J. , Kan F. W. K. , Granger L. , Raymond L. , Bleau G. , Roberts K. D.
Published: February 2005

The nature of cholesterol‐binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [3H]cholesterol and of [3H]cholesteryl sulfate from labeled spermatozoa. Efflux of [3H]sterols was stimulated when the labeled gametes were incubated in Ham's F‐10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density‐gradient ultracentrifugation for lipoprotein analysis, both [3H]cholesterol and [3H]cholesteryl sulfate were found to be carried by very‐low‐density lipoproteins (VLDL), low‐density lipoproteins (VLDL), high‐density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [3H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris‐HCl, 0.56% NaCl, pH 7.8); efflux of [3H]cholesterol from labeled gametes progressed in a time‐dependent manner, but was low in the absence of serum components. The [3H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [3H]sterol binding capacity that is 2—3‐fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.
Effects of lloprost, a stable prostacyclin analog, PGE2 and PGF2α on rabbit blastocysts
Authors: Jones Marjorie A. , Harper Michael J. K.
Published: February 2005

The uptake of prostaglandins (PGs) into day‐6 rabbit blastocysts was studied. Uptake of PGE2 and PGF2α was temperature sensitive and nonsaturable up to 1,000 nM external concentration. In contrast, uptake of Iloprost, a stable prostacyclin analog, was unaffected by temperature, and showed apparent saturation at >2 nM concentration. Individual blastocysts exposed to 0.2 μM radioinert prostaglandins for 1 hr did not swell significantly. Blastocyst concentration of PGF2α was 4.8 ± 2.1‐fold higher than that of PGE2, which was 9.5 ± 2.4‐fold greater than that of 6‐keto‐PGF1α (the stable metabolite of prostacyclin). Uptake of tritiated water was increased by exposure of blastocysts to 0.2 μM PGE2 or PGF2 or PGF2α, but not to Iloprost. Thus, imbibition of water does not cause blastocyst swelling at least during limited in vitro culture. It is concluded that prostacyclin is of less importance in blastocyst function than PGE2 or PGF2α.
Cytogenetic study of parthenogenetically activated bovine oocytes matured in vivo and in vitro
Authors: King W. A. , Xu K. P. , Sirard M.‐A. , Greve T. , Leclerc P. , Lambert R. D. , Jacques P.
Published: February 2005

Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6–8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre‐rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre‐MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre‐MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre‐MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre‐MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.
Fertilization cone formation in starfish oocytes: The role of the egg cortex actin microfilaments in sperm incorporation
Authors: Kyozuka Keiichiro , Osanai Kenzi
Published: February 2005

The process of sperm incorporation into starfish (Asterias amurensis) oocytes was examined by electron and fluorescence microscopy. The fertilization cone began to form at the place where the acrosomal process fused with the egg surface and developed into an inverted conical mass containing a small amount of electron‐dense cytoplasm. Microfilaments, which stained with NBD‐phallacidin, were detected in the fertilization cone. Microvillar protrusions from the fully grown fertilization cone engulfed the sperm head outside the fertilization membrane. The sperm organelles were incorporated into the egg cortex with the absorption of the protrusions. Cytochalasin B inhibited sperm incorporation, fertilization cone formation, and actin filament organization. It is suggested that the development and reduction of the fertilization cone, which depend on the functioning of microfilaments, are necessary for sperm incorporation in starfish.
Characterization of the postacrosomal sheath of bovine spermatozoa
Authors: Olson Gary E. , Winfrey Virginia P.
Published: February 2005

A purified head fraction was prepared from bovine epididymal spermatozoa and was utilized to identify the solubility characteristics and major polypeptide components of the postacrosomal sheath. Sperm heads extracted in nonionic‐detergent‐containing or high‐salt‐containing solutions retained an intact postacrosomal sheath, but it was readily solubilized by high pH extraction solutions. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed a major polypeptide of 58,000 daltons (58‐kD) in the high pH extract solution. Antibodies to the 58‐kD polypeptide specifically reacted with the postacrosomal segment by immunofluorescence and by electron microscopic immunohistochemistry were shown to bind the postacrosomal sheath. We conclude that this 58‐kD polypeptide is a constituent of the postacrosomal sheath and that its distribution is restricted to the postacrosomal segment.
The acrosome reaction in spermatozoa of the grey‐headed flying fox (Pteropus poliocephalus: Chiroptera) exposes barbed subacrosomal material
Authors: Cummins J. M. , Robson S. K. , Rouse G. W.
Published: February 2005

Acrosome reactions were induced in seminal spermatozoa of the megachiropteran Pteropus poliocephalus, the grey‐headed flying fox, using the ionophore A23187 (10 μM in Tyrodes solution with 1% w/v BSA). Samples were taken at 0, 10, and 120 minutes and fixed for electron microscopy. Initial motility of approximately 70% decreased to approximately 10% over 2 hours. The first motile spermatozoa without acrosomal caps were seen after 6 minutes, and by 90 minutes approximately 60% of motile spermatozoa had completed the acrosome reaction. The first sign of the reaction (by TEM) appears to be swelling and cavitation of the acrosomal matrix within the apical and principal segments, followed or possibly accompanied by fusion and fenestration of the outer acrosomal and plasma membranes and dispersion of the matrix except from the equatorial segment. The end‐product of the reaction thus conforms to that seen in other eutherian mammals. However, the reaction exposes a uniquely moulded organization of subacrosomal material (“pseudoperforatorium”), shaped like an anvil over the rostral rim of the flattened nucleus and encased by the remaining inner acrosomal membrane. This has two “lateral” spikes on each side that extend approximately to the same width as the head in the region of the equatorial segment. While these barbs could serve to stabilize or anchor the apical segment of the acrosome, their exposure following the acrosome reaction suggests some specific mechanical role in penetration of the zona pellucida or in protecting more caudal structures from abrasive damage.
Cleavage characteristics of mouse embryos inseminated and cultured after zona pellucida drilling
Authors: Talansky Beth E. , Gordon Jon W.
Published: February 2005

The effects of zona drilling on mouse embryo development in vitro were evaluated. Following insemination, sperm were immediately concentrated at the area of drilling, and in zona‐drilled eggs, pronuclei appeared 30–50 min earlier than in zona‐intact controls. Zona‐drilled oocytes fertilized at significantly higher rates than undrilled controls and, consequently, a greater percentage of eggs inseminated after zona drilling reached the blastocyst stage. The attrition rates of zona‐drilled embryos at each cleavage stage did not differ significantly from controls.

Manipulated embryos exhibited unique cleavage patterns. Some embryos lost their zonae entirely, whereas others became partially extruded at early cleavage stages. These anomalies led to separation of blastomeres from the zygote proper, aggregation of embryos to form giant composite morulae and blastocysts, and occasionally to formation of miniature twin blastocysts. These characteristics of cleavage indicate that although zona drilling of a cohort of oocytes is likely to lead to an increased number of live births relative to controls, some developmental abnormalities can be encountered, and these may be associated with embryo loss, spontaneous chimerism, or possibly with conception of monozygotic twins.
Xenopus spermatozoon: Is there any correlation between motility and oxygen consumption?
Authors: Bernardini Giovanni , Belgiojoso Pio , Camatini Marina
Published: February 2005

The motility status of Xenopus laevis spermatozoa does not affect their respiration rate. Oxygen consumption for 109 spermatozoa is approximately 0.4 μmol/minute. Oxygen consumption is not increased by gramicidin D, an uncoupler, and it is not blocked by KCN or NaN3. The adenosine triphosphate (ATP) content of spermatozoa that have been activated is definitely less than that in the spermatozoa that remained immotile. Incubation in KCN, NaN3, and gramicidin decreases the ATP content and impairs motility. The conclusions of the present study are that in Xenopus spermatozoa motility and oxygen consumption are not correlated, and the composition of the respiratory chain of these spermatozoa presents noteworthy peculiarities.
Segregation products of male mice doubly heterozygous for the RB(6.16) and RB(16.17) translocations: Influence of sperm karyotype on fertilizing competence under varying mating frequencies
Authors: Zackowski Joleen L. , Martin‐DeLeon Patricia A.
Published: February 2005

The meiotic segregants of male mice heterozygous for Rb(6.16)24Lub and Rb(16.17)7Bnr were viewed, for the first time, at first cleavage metaphase. Chromosomes were analyzed after G‐banding, C‐banding, and karyotyping. To study sperm aging effects, chromosomes of 202 one‐cell zygotes derived from males mating at intervals of approximately 3,14, and 21 days were examined. At least 89.6% of sperm‐derived complements were products of 2:2 segregation; at most, a possible 6.4% were 3:1 segregants. The six expected types of 2:2 segregants, both balanced and unbalanced, were equifrequent in the total zygote population derived from sperm of all ages. When the data were analyzed according to mating frequency, the 3‐day sperm population considered most likely to be fresh showed a deficiency of the segregant nullisomic for chromosome 6 and disomic for chromosome 17, when compared with the reciprocal segregant (P < 0.025) as well as to all other 2:2 segregants (P < 0.05). However, these sperm fertilized in greater numbers (P < 0.01) than their reciprocal segregant (disomic for 6 and nullisomic for 17) in the 14‐day sperm population. While sperm with chromosomal abnormalities are capable of fertilization, the competence of segregants nullisomic for 6 and disomic for 17 apparently depends on the prior storage period in the male. Further, the results suggest that the effect of aneuploidy on sperm function is dependent on the specific chromosome(s) involved.
Preimplantation mammalian aggregation and injection chimeras
Authors: Prather Randall S. , Hagemann Lora J. , First Neal L.
Published: February 2005

The preimplantation embryo is highly resilient to experimental manipulations. A specific manipulation that has revealed many clues to the developmental process is chimera production. Chimeras have been used to describe the importance of developmental characteristics of embryonic cells and how these characteristics are involved with developmental fate. These characteristics have been monopolized in the production of interspecific chimeras and the production of transgenic animals. This review attempts to discuss the major factors affecting preimplantation mammalian embryo chimera production.
Effects of ultrasound on ovulation in the mouse
Authors: Heyner S. , Abraham V. , Wikarczuk M. L. , Ziskin M. C.
Published: February 2005

In vitro fertilization and embryo transfer (IVF–ET) programs use ultrasound extensively for monitoring the growth of ovarian follicles and, subsequently, for confirming the presence of a fetal sac. There have been few reports of the effects of ultrasound on ovulation rates in mammals, and we report here that following exposure to continuous wave ultrasound at a spatial average intensity of 3.0 W/cm2 for five minutes, ovulation rates measured 10 days later were significantly reduced in mice. When temperature elevation of the exposed ovary was measured with a thermocouple, hyperthermia correlated with reduction in ovulation.
Development of single blastomeres from four‐ and eight‐cell mouse embryos fused into the enucleated half of a two‐cell embryo
Authors: Kono T. , Tsunoda Y.
Published: February 2005

Single blastomeres from four‐ and eight‐cell mouse embryos were fused into the enucleated halves of two‐cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four‐cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight‐cell embryo blastomeres were used. Eight complete sets of the quadruplet‐reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight‐cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four‐cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight‐cell embryos lacked an ICM. These results suggest that the genomes of four‐ and eight‐cell nuclei introduced into the enucleated halves of two‐cell embryos are reversed to support the development of the reconstituted embryo.
Sperm morphology and storage in the female reproductive tract of the fat‐tailed dunnart, Sminthopsis crassicaudata (Marsupialia: Dasyuridae)
Authors: Breed W. G. , Leigh C. M. , Bennett J. H.
Published: February 2005

A study of spermatozoa in the isthmus of the oviduct and of the surrounding epithelial cells in the dasyurid marsupial, Sminthopsis crassicaudata, was carried out. At least 10% of the ejaculated spermatozoa probably populate the isthmus region, where many come to reside in crypts until around the time of ovulation. Ultrastructural observations of spermatozoa in this region indicated that they had intact acrosomes and were identical in their morphology with those in the cauda epididymidis. After ovulation spermatozoa rapidly disappeared, some of which may be phagocytosed by the cells lining the crypts. These epithelial cells were also found to have many large, electron‐dense granules at the time of sperm storage, but the contents did not appear to be released until the zygotes passed along the tract. The secretory activity of these cells may thus relate more to the production of the shell membrane that comes to surround the zygote than to the cells performing a nutritive or protective function for the spermatozoa during their period of storage within the female reproductive tract.
Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation
Authors: Alvarez Juan G. , Storey Bayard T.
Published: February 2005

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H2O2. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H2O2 on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1‐5 mM H2O2, while the rate in rabbit sperm is unaffected by H2O2. The enhancement of lipid peroxidation, the rate of reaction of H2O2 with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H2O2 due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H2O2. This implies that H2O2 by itself at 1‐5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H2O2 or mercapto‐succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O2 as the rate‐determining step.
Characterization of cell surface polypeptides of unfertilized, fertilized, and protease‐treated zona‐free mouse eggs
Authors: Boldt Jeffrey , Gunter L. E. , Howe Anita M.
Published: February 2005

The polypeptide composition of unfertilized, fertilized, and protease‐treated zona‐free mouse eggs was evaluated in this study. Zona‐free eggs were radioiodinated by an Iodogen‐catalyzed reaction. Light microscopic autoradiography of egg sections revealed that labeling was restricted to the cell surface. Labeled eggs were solubilized, and cell surface polypeptides were identified by one‐dimensional SDS polyacrylamide gel electro‐phoresis and autoradiography. The unfertilized egg demonstrated 8–10 peptides that incorporated125I, with major bands observed at approximately 145–150, 94, and 23 kilo‐daltons (kD). Zona‐free eggs fertilized in vitro and then radiolabeled demonstrated several new bands in comparison to unfertilized eggs, with a major band appearing at approxi mately 36 kD. Treatment of radiolabeled unfertilized eggs with either trypsin or chymo‐trypsin (1 mg/ml for 5–20 min) caused enzyme‐specific modifications in labeled polypep tides. Trypsin (T) treatment resulted in time‐dependant modification of the three major peptides at 145–150,94, and 23 kD. Chymotrypsin (CT) treatment, in contrast, was asso ciated with loss or modification of the 94 kD band, with no apparent effect on either the 145–150 or 23 kD band. Taken together with previous data indicating that T or CT egg treatment interferes with sperm‐egg attachment and fusion (Boldt et al.: Biol Reprod 39:19–27, 1988), these results suggest a possible role for the 94 kD protein in sperm‐egg interaction.
In vitro fertilization in the sheep: Effect of elevated calcium concentration at insemination
Authors: Huneau D. , Crozet N.
Published: February 2005

Oocytcs (n = 273), collected from superovulated ewes, were inseminated with in vitro capacitated spermatozoa from four rams [Crozet et al., 1987]. In each experiment, paral lel insemination was performed using aliquants from a single ejaculate in either our stan‐dard fertilization medium DM‐H‐SS (a modification of Bracken's defined medium, buffered with HEPES and containing 20% v/v sheep serum) or in the same medium supplemented with calcium lactate (DM‐H‐SS + Ca). The measured total calcium con centrations were Ca++T = 2.74 mM in DM‐H‐SS medium and Ca++T = 8.74 mM in DM‐H‐SS + Ca; the ratio of free to total calcium in DM‐H‐SS was Ca++F/Ca++T = 0.85. Fertilization was assessed at 17 hours postinsemination.

Variations in the ejaculates were observed for each of the four rams tested. When DM‐H‐SS + Ca was used, the percentages of fertilized (75% vs. 50%) and monospermic (58% vs. 41%) eggs were significantly enhanced compared with use of DM‐H‐SS. No improve ment was observed in control medium DM‐H‐SS + lac containing neutralized lactic acid. Supplementing the fertilization medium with calcium had no apparent effect on the inci dence of polyspermy.

These experiments show that the fertilization rate achievable in vitro by individual ejaculates from various rams can be increased by raising the calcium concentration in the fertilization medium to a value much higher than that present in tuba! fluids from estrous ewes. Extended incubation in such a high calcium concentration is unnecessary for in vitro capacitation of ram spermatozoa.
Primordial germ cell development in cultures of dispersed central disks of stage X chick blastoderms
Authors: Ginsburg Malka , Eyal‐Giladi Hefzibah
Published: February 2005

Central disk fragments cut from stage X chick blastoderms were dispersed and cultured on glass coverslips. After 48 hr of incubation the cultures showed various degrees of organization into three‐layered aggregates in which no axis development was observed. Primordial germ cells (PGCs) were detected in ail cultures. The number of PGCs was found to be correlated to the initial cell concentration in the suspension. By regression analysis it was found that in cultures initiated from 10 central disks or more, the mean number of PGCs per fragment was constant and matched the number of PGCs found for intact control central disks incubated for the same length of time. It appears that in cultures of stage X, the morphologic expression of PGCs is related to the level of differentiation and organization of the somatic cells in the culture, which, in turn, is dependent on the initial concentration of cells in the culture.
Ultrastructure of spermiogenesis in the gastropod Calliotropis glyptus Watson (Prosobranchia: Trochidae)with special reference to the embedded acrosome
Authors: Healy John M.
Published: February 2005

Testicular spermatozoa and sperm development in the archaeogastropod Calliotropis glyptus Watson (Trochoidae: Trochidae) are examined using transmission electron microscopy and formalin‐fixed tissues. During spermiogenesis, the acrosome, formed evidently through fusion of Golgi‐derived proacrosomal vesicles, becomes deeply embedded in the condensing spermatid nucleus. Two centrioles (proximal and distal), both showing triplet microtubular substructure, are present in spermatids—the distal centriole giving rise to the sperm tail and its associated rootlet. During formation of the basal invagination in the spermatid nucleus, centrioles, and rootlet move towards the nucleus and come to lie totally within the basal invagination. Mitochondria are initially positioned near the base of the nucleus but subsequently become laterally displaced. Morphology of the mature spermatozoon is modified from that of the classic primitive or ect‐aquasperm type by having 1) the acrosome embedded in the nucleus (the only known example within the Mollusca), 2) a deep basai invagination in the nucleus containing proximal and distal centrioles and an enveloping matrix (derived from the rootlet), 3) laterally displaced periaxonemal mitochondria, and 4) a tail extending from the basal invagination of the nucleus. Implantation of the acrosomal complex and centrioles within imaginations of the nucleus and lateral displacement of mitochondria effectively minimize the length of the sperm head and midpiece. Such modifications may be associated with motility demands, but this remains to be established. The unusual features of C. glyptus spermatozoa, though easily derivable from ‘typical’ trochoid sperm architecture, may prove useful in delineating the genus Calliotropis or tracing its relationship to other genera within the trochid subfamily Margaritinae.
Trout sertoli cells and germ cells in primary culture: I. Morphological and ultrastructural study
Authors: Loir Maurice
Published: February 2005

In order to characterize trout Sertoli cells and germ cells obtained after testis dissociation and cell separation, we have studied their morphology, ultrastructure, survival, and ability to express differentiated activities in primary cultures. After dissociation, the fine structure of Sertoli cells does not differ from that observed in situ and only minor changes are shown for at least 13 days. Until they are flattened in a monolayer, they keep the ability to retain germ cells on their surface. When flattened, some of them are able to divide. At the opposite of meiotic germ cells, spermatogonia can develop independently of Sertoli cells. They are able to proliferate during at least 10 days. Spermatocytes and spermatids are obtained as single cells and multinucleated giant cells (symplasts). In the absence of somatic cells, their maximal viability is approximately 5 days, whereas spermatocytes adhering to Sertoli cells can survive at least 10–12 days, provided trout lipoproteins are present. Spermatocytes are able to differentiate to spermatids, although this process is impaired for some ceils. The adhesion of spermatogonia and spermatocytes to Sertoli cells is specific, mediated by desmosome‐like junctions and favored by lipoproteins. These data are compared to what is known in mammals and in amphibians.
Adrenergic stimulation of sea urchin sperm cells
Authors: Nelson Leonard , Cariello Lucio
Published: February 2005

The noradrenergic agonists norepinephrine and isoproterenol elicit greater stimulatory swim ming responses in sea urchin spermatozoa than epinephrine. The β‐blocker atenolol induces an even greater motile rate, while the α‐blocker phentolamine has only a moderate effect, it also causes a minimal reduction in the sperm cells' response to atenolpol. Caffeine increases the motility but to a lesser degree than 8‐Br‐cAMP. In drug interaction assays, both caffeine and 8‐Br‐cAMP depress the adrenergic effects. Agents that affect access of calcium to the flagellar apparatus (verapamil and trifluoperazine) depress the motility below the level of the controls when incubated separately with the sperm suspensions and counteract the stimulation due to atenolol. Adrenergic modulation of sperm motility thus appears to be both a calcium‐dependent and a cyclic nucleotide‐dependent process.
Lectin binding characterstics of mouse epididymal fluid and sperm extracts
Authors: Rankin Tracy L. , Holland Michael K. , Orgebin‐Crist Marie‐Claire
Published: February 2005

Glycoproteins from luminal fluid of the mouse cauda epiciidymidis have been compared with glycoproteins from Triton X‐100 extracts of mouse spermatozoa from varying regions of the epididymis, using lectins with specific affinity for different sugar residues. Concanavalin A recognizes 11 glycocomponents on Western blots of fractionated caudal fluid; wheat germ agglutinin (WGA) binds 12 proteins; Ulex europaeus agglutinin (UEA) binds seven; and Dolichos biflorus agglutinin (DBA) recognizes nine. Several of these glycoproteins display an affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues; whereas other proteins bind only one of the four lectins used. The results also show that some glycoproteins exhibit a higher affinity for particular lectins. Eight glycoproteins of similar mobility and lectin‐binding characteristics are detected in Triton X‐100 extracts of spermatozoa from different regions of the epididymis and in caudal fluid. The lectin affinity of some proteins appears or increases in spermatozoa from distal epididymal regions (54 kD, 32 kD), whereas the lectin affinity of others decreases (29 kD, 40 kD). There are differences in lectin affinities between proteins in sperm extracts and in caudal fluid. Some proteins show an affinity for three or four lectins in caudal fluid, but proteins of similar electrophoretic mobility in sperm extracts bind only one or two of the lectins. These data show that glycoproteins of similar mobility are present in caudal fluid and in Triton‐X‐100 sperm extracts, implying a potential interaction between caudal fluid components and epididymal sperm.
In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension
Authors: Hamano S. , Naito K. , Fukuda Y. , Toyoda Y.
Published: February 2005

The fertilizing ability of boar ejaculated spermatozoa was examined in vitro after prcincubation at a concentration of 2.5 × 108/ml for 4 hr in several conditioned media (CM). For preparation of CM, boar spermatozoa were incubated in a modified Krebs‐Ringer bicarbonate solution (TYH) at concentrations of 20 to 40 × 108/ml for several hours up to 4 hr; then their supernatant fluids were collected by centrifugation. When boar ejaculated spermatozoa were preincubated in TYH alone, 14.1% of oocytes were penetrated by them as we reported previously. On the other hand, preincubating them with CM, their fertilizing ability was elevated according as the incubation time of CM preparation was lengthened. The fertilization rate reached 75.0%, using 4 hr‐incubated CM for the preincubation medium. The effect of CM was not deteriorated by heat treatments (56°C, 30 min, or 100°C, 5 min). The components of CM were separated at a molecular weight of 25,000 by ultrafiltration, and high fertilization rate (69.8%) was obtained when low molecular weight fraction was used for the preincubation medium. Sperm extracts prepared from directly frozen‐thawed sperm suspension and 0.1–10 mM of taurine or hypotaurine had no effect on the fertilizing ability of boar spermatozoa. These results suggest that substances stimulating boar sperm capacitation were accumulated from viable spermatozoa into the medium during incubation and that the effective substances were heat‐stable and of low molecular weight and were not taurine and hypotaurine.
Interval estimation of the difference in proportions under m‐to‐one matching
Authors: Lui Kung‐Jong
Published: February 2005

When the number of potential controls is large relative to the number of available cases, or when little effort needs to be expended in collecting the relevant information on the controls, we often apply multiple matching to attain the validity or increase the efficiency of our inference in epidemiological studies. In this paper, we focus interval estimation on the difference in proportions for m‐to‐one matching. We consider four asymptotic interval estimators, including the estimator directly using the Mantel–Haenszel (MH) point estimator, the estimator using the tanh−1(x) transformation, the estimator derived from the Cochran–Mantel–Haenszel (CMH) test statistic, and the estimator derived from the quadratic inequality developed in this paper. To evaluate and compare the performance of these estimators, we employ Monte Carlo simulation. We find that the estimator directly using the MH estimator can have the coverage probability less than the desired confidence level when the number of matched sets is small. We note that the estimator derived from the quadratic inequality can perform well when the underlying difference is close to 0 even for a small number of matched sets. However, this estimator tends to have the coverage probability less than the desired confidence level as well when the underlying difference in proportions is large. By contrast, the estimator using the CMH statistic tends to have the coverage probability larger than the desired confidence level when the underlying difference is small. We also find that the estimator using the tanh−1(x) transformation consistently outperforms the interval estimator directly using the MH estimator. We use the data regarding the association between induced abortions and ectopic pregnancy to illustrate the use of these estimators. Copyright © 2005 John Wiley & Sons, Ltd.
Single‐Walled Carbon Nanotubes Under the Influence of Dynamic Coordination and Supramolecular Chemistry
Authors: Chichak Kelly S. , Star Alexander , Altoé M. Virginia P. , Stoddart J. Fraser
Published: February 2005

A dynamic coordinative‐directed solubilization of single‐walled carbon nanotubes (SWNTs) in aqueous solutions has been achieved through a combination of a ZnII metalloporphyrin complex and a cis‐protected PdII complex, which are believed to form charged acyclic and/or cyclic adducts on or around the side walls of SWNTs. The solubilization of SWNTs in aqueous solution only occurs when these acyclic and/or cyclic complexes are allowed to enter simultaneously into a self‐assembly process with SWNTs under mild conditions. The aqueous solubility properties that these dynamic complexes confer upon SWNTs are believed to involve noncovalent bonding interactions between the two entities. They have been probed in solution using ultraviolet and visible absorption spectroscopy and in thin films using high‐resolution transmission electron microscopy. The supramolecular electronic effects that the individual components of their acyclic and/or cyclic complexes impart upon a single semiconducting SWNT have been probed within a nanotube field‐effect transistor device.
Noble Metal Nanoparticles Deposited on Self‐Assembled Monolayers by Pulsed Laser Deposition Show Coulomb Blockade at Room Temperature
Authors: Speets Emiel A. , Dordi Barbara , Ravoo Bart Jan , Oncel Nuri , Hallbäck Ann‐Sofie , Zandvliet Harold J. W. , Poelsema Bene , Rijnders Guus , Blank Dave H. A. , Reinhoudt David N.
Published: February 2005

Nanometer‐sized noble‐metal clusters are fabricated on top of alkylthiolate self‐assembled monolayers (SAMs) on annealed gold by pulsed laser deposition at elevated pressures. The size distribution of the clusters depends on the metal and on the pressure during the deposition. Scanning tunneling microscopy (STM) and conductive probe atomic force microscopy (CP‐AFM) showed that the metal clusters are insulated from the substrate on top of the SAM. Coulomb blockades could be measured at room temperature by STM for palladium clusters on decanethiol SAMs.
Carbon Nanotubes for Microelectronics?
Authors: Graham Andrew P. , Duesberg Georg S. , Seidel Robert V. , Liebau Maik , Unger Eugen , Pamler Werner , Kreupl Franz , Hoenlein Wolfgang
Published: February 2005

Despite all prophecies of its end, silicon‐based microelectronics still follows Moore's Law and continues to develop rapidly. However, the inherent physical limits will eventually be reached. Carbon nanotubes offer the potential for further miniaturization as long as it is possible to selectively deposit them with defined properties.