bluuebunny/crossref_metadata_2025_split huggingface dataset:
The distribution of organelles in mammalian oocytes following centrifugation prior to injection of foreign DNA
Authors: Cran D. G. 
Published: February 2005

AbstractThe centrifugation of oocytes from the domestic species is a necessary prerequisite to allow visualization of nuclei for the introduction of foreign DNA. This improvement in visibility which has allowed the production of transgenic animals is accompanied by a clear stratification of the organelles. In immature oocytes from the sheep, pig, and cow, four distinct zones are formed. These comprise, lipid, membrane‐bound vesicles, organelle‐free cytoplasm, and mitochondria. In mature ovine oocytes, a fifth zone of smooth endoplasmic reticulum (SER) beneath that of the vesicles is formed. This SER is produced at discrete locations within the untreated cell. The potential for removal of these fractions has implications for relating patterns of protein synthesis with particular structural components.In intact oocytes, the cortical granules are located in a peripheral position beneath the plasma membrane. However, even after subjection to high centrifugal force (65,000g for 60 min), they maintain their original location. However, treatment with the cytoskeletal inhibitors, nocodozole and cytochalasin, results in rapid exocytosis after centrifugation. It is concluded that the maintenance of the spatial relationships of this organelle is mediated through the peripheral cytoskeleton.
DNA synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes
Authors: Naish Sally J. , Perreault Sally D. , Zirkin Barry R. 
Published: February 2005

AbstractWe have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H‐thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. We conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.
Effects of in utero and in vitro incubation on the lipid‐bound fatty acids and sterols of porcine spermatozoa
Authors: Evans R. W. , Weaver D. E. , Clegg E. D. 
Published: February 2005

AbstractSperm‐rich semen and washed porcine spermatozoa were incubated for up to 2 hr either in utero in the presence of oviduct fluid or in vitro at 37°C. Sperm lipids were extracted and separated into phospholid and neutral lipid fractions. Eleven phospholipid and five neutral lipid fatty acids were identified and quantified using GC and GC‐MS. The percentage of 22:5n6, the major phospholipid fatty acid, decreased slightly but significantly during 1.5 hr of in utero incubation (41.2–38.0%), but after 2.0 hr of in utero incubation no significant difference was observed (40.0%). None of the phospholipid fatty acids changed in concentration during in vitro incubation. The mole ratio of phospholipid to phospholipid fatty acid (1.00:1.27) did not change during incubation. The levels of neutral lipid‐bound 14:0 decreased (43.5% to 31.8%) and that of 18:0 increased (11.1% to 18.2%) during in utero incubation. Similar but less pronounced changes were observed during in vitro incubation. (43.5% to 36.0%; and 11.1% to 15.8%, respectively).Two major sterols, cholestrol (73%) and desmosterol (27%) were identified by gas chromatography–mass spectrometry. The mole ratio of phospholipid to sterol (2.47:1:00) did not change during incubation.
Zona pellucida penetration assay for capacitation of bovine sperm
Authors: Wheeler Matthew B. , Seidel George E. 
Published: February 2005

AbstractIn vitro capacitation and in vitro zona penetration systems are described for bovine spermatozoa. The criterion for capacitation was penetration by sperm of either 1) the zona pellucida of dead bovine oocytes, or 2) live, intact oocytes in which subsequent development was observed. Sperm were washed free of seminal plasma by centrifugation and incubated in a modified Tyrode's medium (TALP) in a 5% CO2‐air atmosphere at 38°C. Dead bovine oocytes were obtained from > 1 mm follicles from ovaries stored at −20°C. Spermatozoa at a final concentration of 107 ml were coincubated with the oocytes for 3 hr after capacitation treatments.Initial experiments established that: 1) dead bovine oocytes can be penetrated by bovine sperm capacitated in the rabbit reproductive tract; 2) oocytes should be used for the penetration test within 24 hr of thawing; 3) oocytes could be stored for 24 hr at 4°C after penetration before evaluation; and 4) penetration of zonae pellucidae of dead bovine oocytes by sperm is species specific.The percentages of dead oocytes penetrated after incubating frozen‐thawed sperm for 0, 6, 12, 24, and 30 hr prior to incubation with oocytes were 17, 24, 34, 10, and 0% respectively; the corresponding percentages for fresh semen were 1, 2, 5, 42, and 25%. Addition of 1% bovine seminal plasma to suspensions of washed, capacitated sperm decreased oocyte penetration percentages from 48% for the control (no seminal plasma) to 23%.Spermatozoa capacitated in vitro for 24 hr fertilized 15 of 22 ovulated oocytes (68%) from superovulated cows. Three embryos developed to the morula stage, but no pregnancy resulted after transfer of these embryos to bovine recipients.The present results provide the basis for a reliable (interassay C.V. = 13.4%) in vitro assay system for capacitation based on sperm penetration of the zona pellucida of dead bovine oocytes.
Membrane molecules involved in adhesion properties of cultured sertoli cells
Authors: Zani Bianca M. , Ziparo Elio , Russo Mario A. , Filippini Antonio , Stefanini Mario 
Published: February 2005

AbstractMembrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody‐induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS‐PAGE), Mr 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.
A cortical granule‐specific enzyme, B‐1,3‐glucanase, in sea urchin eggs
Authors: Wessel Gary M. , Truschel Margaret R. , Chambers Scott A. , McClay David R. 
Published: February 2005

AbstractThe ultrastructural localization of B‐1,3‐glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B‐1,3‐glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B‐1,3‐glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.
Immunological approach to characterize proacrosin and various acrosin forms in boar and man by monoclonal antibodies
Authors: Kaufmann M. , Schönwald D. , Mansouri A. , Günther E. 
Published: February 2005

AbstractThree different monoclonal rat antibodies, Acr1, Acr2, and Acr3, have been established against boar proacrosin. They are shown by enzyme‐linked immunosorbent and immunoblot assays to react with boar proacrosin and several different acrosin molecules derived therefrom during activation. The epitopes detected by the three antibodies are different from each other, one being highly sensitive to reduction and periodate treatment. The antibodies crossreact with various proacrosin and acrosin molecules derived from human sperm extract; they also show indirect immunofluorescent staining of the acrosomal region of ejaculated sperm from normal men but fail to react with round‐headed spermatozoa.
Correlation between sperm creatine phosphokinase activity and sperm concentrations in normospermic and oligospermic men
Authors: Huszar Gabor , Corrales Marcelia , Vigue Lynne 
Published: February 2005

AbstractToward the development of biochemical probes for the assessment of sperm function we have measured the activities of sperm creatine‐N‐phosphotransferase (CPK). There was a highly significant inverse correlation (P < 0.001 in all comparisons) between sperm CPK activities and sperm concentrations in specimens of normospermic and oligospermic men with > 30 million sperm/ml (0.106 ± 0.01 SEM, Nequals;90, expressed as CPK U/100 million sperm), 20–30 million sperm/ml (0.333 ± 0.07 SEM, Nequals;30) and 10–20 million sperm/ml (0.583 ± 0.12 SEM, Nequals;30) when compared with the CPK values of the < 10 million/ml specimens (2.242 ± 0.46 SEM, Nequals;30). Furthermore, the distribution of CPK activities within these four groups showed that 96%, 67%, 43%, and 4% of the samples, respectively, were in the < 0.250 CPK U/100 million sperm normal range (mean + 2 SD of the > 30 million sperm/ml group). However, there was no relationship between sperm CPK activities and the values of sperm motility (P > 0.15) or morphology (Pequals;0.38) in the samples. The migrated sperm fractions (significantly improved in motility and velocity parameters) showed CPK activities lower than the initial semen specimens (P < 0.01, Nequals;150). In fact, in some oligospermic men the CPK activities of the migrated sperm fractions were within the range of normospermic samples. The data suggest that sperm CPK values in the initial specimens and the degree of improvement in the migrated sperm fractions reflect the relative concentrations of a “normal” sperm subpopulation. We propose that CPK activities and similar objective biochemical parameters may be important in predicting sperm quality and the fertilizing potential of oligospermic men.
Ultrastructure of the sperm and spermatogenesis and spermiogenesis of Dina lineata (hirudinea, erpobdellidae)
Authors: Bonet S. , Molinas M. 
Published: February 2005

AbstractThe 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

AbstractPrevious 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

AbstractThe 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

AbstractThe 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

AbstractPrevious 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

AbstractThe 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

AbstractThe 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

AbstractThe 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.