bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence In situ hybridization to Y chromosomes in decondensed human sperm nuclei
Authors: Wyrobek Andrew J. , Alhborn Thomas , Balhorn Rod , Stanker Larry , Pinkel Daniel 
Published: February 2005

AbstractHuman sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome‐specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.
Co‐culture of rabbit 2‐Cell embryos with rabbit oviduct epithelial cells and other somatic cells
Authors: Carney E. W. , Tobback C. , Ellington J. E. , Foote R. H. 
Published: February 2005

AbstractRabbit 2‐cell embryos were co‐cultured in Basal Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast‐like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39°C in 5% CO2in air, 77–93% of the rabbit embryos which were co‐cultured with somatic cells had reached the blastocyst stage, and 60–76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P>.05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239–302 μm). Bovine 1–8‐cell embryos were also co‐cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so‐called “16‐cell block” in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow. However, other factors besides those provided by the ROEC cultures must also be important for optimal development, as both rabbit and bovine embryos develop more slowly in co‐culture than in vivo.
Early embryo development in the Siberian hamster (Phodopus sungorus)
Authors: Nieder Gary L. , Caprio Terri L. 
Published: February 2005

AbstractDevelopment of preimplantation embryos of the Siberian hamster (Phodopus sungorus) in vivo and in vitro was examined. The timing of early development in vivo was found to be slower than that reported for the golden hamster. Progression through the cleavage stages, cavitation, and hatching from the zona pellucida occurred later, with blastocyst formation beginning on the afternoon of day 4 and uterine attachment occurring early on day 5. In vitro, morulae, and early blastocysts collected on day 4 and cultured in serum‐containing medium formed expanded blastocysts and some began to hatch from the zona pellucida. With extended culture, blastocysts attached and formed trophoblast outgrowths. Outgrowth was characterized by an initial migration of small cells from the blastocyst, followed by formation of a sheet of trophoblast giant cells. Differences in the morphology of outgrowth between the hamster and mouse suggest that further comparative studies with the Siberian hamster may be useful.
Nonisotopic in situ hybridization as a method for nondisjunction studies in human spermatozoa
Authors: Coonen Edith , Pieters Math H. E. C. , Dumoulin John C. M. , Meyer Henk , Evers Johannes L. H. , Ramaekers Frans C. S. , Geraedts Joep P. M. 
Published: February 2005

AbstractHuman spermatozoa were studied with a nonradioactive in situ hybridization method. Using a chemically modified DNA probe and immunocytochemical reactions for visualization, it was possible to obtain hybridization signals in 31 of 32 semen samples. Positive hybridization reactions, depending on cell accessibility, varied from 40% to over 90% for the different samples. Using a chromosome 1‐specific DNA probe, disomy for this chromosome was found in 0.67% of all accessible sperm cells.
Expression and activity of gamma‐glutamyl transpeptidase in the rat epididymis
Authors: Hinton Barry T. , Palladino Michael A. , Mattmueller Danette R. , Bard Dawn , Good Kyle 
Published: February 2005

AbstractFollowing Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ducts deferens were 2.2, 2.3, 2.2., and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput=corpus>cauda and corpus>caput>cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine‐borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 μM and 2.19 nmoles/min/μl luminal fluid at pH 8.5 compared to 0.49 μM and 0.49 nmoles/min/μl luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 μM) and higher Vmax (1.13 nmoles/min/μl luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X‐114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions. Epididymal GGT may not be involved in the transport of L‐glutamate since transport was not related to the degree of GGT mRNA expression along the epididymal duct.
Identification of spectrin and calmodulin in rabbit spermiogenesis and spermatozoa
Authors: Camatini Marina , Colombo Anita , Bonfanti Patrizia 
Published: February 2005

AbstractActin was localized in the four subacrosomal bulges (Camatini et al., Eur. J. Cell Biol. 45:276–281, 1987), which characterize the sperm head of rabbit spermatozoa (Phillips, J. Ultrastruct. Res. 38:591–604, 1972), and in the postacrosomal region (Welch and O'Rand, Dev. Biol. 109:411–417, 1985; Camatini et al., Eur. J. Cell Biol. 45:276–281, 1987). Specific antibodies and indirect immunogold labelling on testis Lowicryl K4M sections and spermatozoa cryosections were used to study the distribution of calmodulin and a spectrin‐like protein. This protein was also present close to the shaping membranes of the head. The results presented suggest a membrane‐cytoskeletal role of actin, spectrin, and calmodulin.
Response of porcine oocytes to electrical and chemical activation during maturation in vitro
Authors: Hagen D. R. , Prather R. S. , First N. L. 
Published: February 2005

AbstractThese studies were conducted to examine activation of in vitro‐matured porcine oocytes in response to an electrical stimulus or to an ionophore. Cumulus‐enclosed porcine oocytes were incubated in maturation medium supplemented with either FSH and LH (MM:Exp.1) or pregnant mare serum gonadotropin (PMSG; MMP: experiments 2–4) at 39°C in 5% CO2:95% air with high humidity. In experiment 1, groups of oocytes were stripped of cumulus and then shampulsed (control) or electrically pulsed with a Zimmerman Cell Fusion unit at 24, 31, 41, 48, and 65 h of incubation. Control oocytes were exposed to the activation medium for 20 sec, whereas oocytes to be pulsed were subjected to a single activation pulse (120 V, 30 μsec). Oocytes were cultured for an additional 24 h and then fixed and examined. For oocytes pulsed at 24, 31, 41, 48, and 65 h, the proportions which activated were 0, 0, 87, 88, and 83%, respectively. In experiment 2, oocytes were electrically or sham‐pulsed with a BTX 200 Embryomanipulation System at 24, 30, and 40 h of incubation and respective proportions of oocytes activating were 27%, 39%, and 72%. In experiment 3, oocytes were subjected to 0, 1, or 2 activation pulses after 41 h of incubation in MM‐P. Double‐pulsing halved the proportion of activated oocytes (P<.0001). In experiment 4, oocytes were subjected to 0, 25, 50, or 100 μM ionophore at 48 h of incubation. Proportions of oocytes activated by ionophore were greater than for control (P < .05), but activation was not increased by increasing dose of ionophore. These results indicate that porcine oocytes may respond to an activation stimulus by 24 h of incubation, long before maturation is normally completed. Activation can be achieved also with an ionophore, as described previosly for other species.
Transforming growth factor‐alpha augments meiotic maturation of cumulus cell‐enclosed mouse oocytes
Authors: Brucker Cosima , Alexander Nancy J. , Hodgen Gary D. , Sandow Bruce A. 
Published: February 2005

AbstractGrowth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor‐α (TGF‐α) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell‐enclosed oocytes were exposed to TGF‐α with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denucded oocytes were examined for GVBD following exposure to HX and TGF‐α. When cumulus cell‐enclosed oocytes were exposed to TGF‐α (1 μg/ml) in the presence of HX (4 mM), an increrase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF‐α and HX as compared to 33% with HX only. Concentrations of TGF‐α as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF‐α, but without HX, was also enhanced. The stimulation of GVBD by TGF‐α showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF‐α in the presence of HX, no effect was observed. Our results suggest that TGF‐α is a potent stimulator of mouse oocyte maturation in vitro and that is effect is mediated by the surrounding cumulus cells. In view of the established presence and action of TGF‐α in the intact ovary, these studies indicate that TGF‐α may be part of an intricate system regulating the latter stages of maturation of oocytes during follicular development in vivo.
Spontaneous and induced activation of rat oocytes
Authors: Zernicka‐Goetz Magdalena 
Published: February 2005

AbstractOvulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase‐like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase–‐the two processes that normally occur in succession as a response to oocyte activatin‐–can be experimentally separated.
Synthesis and developmental regulation of an egg specific mouse protein translated from maternal mRNA
Authors: Richoux Veronique , Renard Jean‐Paul , Babinet Charles 
Published: February 2005

AbstractProteins synthesized by DDK mice embryos were analyzed by 2D electrophoresis and a new egg‐specific polypeptide, D14, was identified. The protein is characterized by its high rate of synthesis and electrophoretic properties (MW 36,500, pl>8). The synthesis of D14 is strictly developmentally regulated: starting in the maturing oocyte in the few hours following germinal vesicle breakdown (GVBD), it remains high over the first cell cycle and decreases abruptly during the two‐cell stage. The arrest of D14 synthesis is triggered by egg activation and does not directly depend on transcription by the zygotic genome. Nevertheless, drugs that perturb the onset of zygotic transcription concomittently inhibit D14 arrest of synthesis. D14 is present in both cytoplasmic and nuclear compartments at the two‐cell stage; it is very stable and remains detectable at least until the eight‐cell stage in the preimplantation embryo. Embryos of wild strains of mice synthesized either D14 or a D14 related polypeptide at a rate comparable to that of DDK embryos, which was at least ten times greater than that found in other laboratory strains. Both the developmental regulation and the genetic variability in its rate of synthesis make D14 an interesting polypeptide for the study of regulation of maternal information in the very early stages of mouse embryo development.
Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation
Authors: Bloom Theodora 
Published: February 2005

AbstractCompaction, occurring at the eight‐cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development (TL Bloom, J McConnell: Mol Reprod Dev 26:199–210, 1990). In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with [32P]orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four‐cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four‐cell embryos. Inhibition of protein synthesis at the eight‐cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight‐cell but not in four‐cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight‐cell embryos incubated in phorbol 12‐myristate 13‐acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphorprotein spots associated with the eight‐cell but not with the four‐cell stage. Embryos incubated in Ca2+‐free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.
Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes
Authors: de Loos F. , Kastrop P. , van Maurik P. , van Beneden Th. H. , Kruip Th. A. M. 
Published: February 2005

AbstractThe integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first‐category COC predominantly possessed the penetrating CCPE, the fourth‐category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus‐oocyte contacts was assessed by means of incorporation of 3H‐choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.
Energy metabolism of spermatozoa of the sea urchin Glyptocidaris crenularis
Authors: Mita Masatoshi 
Published: February 2005

AbstractEnergy metabolism in spermatozoa of the sea urchin Glyptocidaris crenularis was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol but also triacylglycerides (TG). Following dilution of the dry sperm in sea water, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for an increase in the level of free fatty acid. Oil red‐O staining of spermatozoa showed that TG was principally located in part of the sperm midpiece. Also, high lipase activity was demonstrated in the spermatozoa. In both intact cells and a cell‐free system, 14C‐labeled fatty acids were oxidized to 14CO2. It is thus concluded that G. crenularis spermatozoa use TG as a substrate for energy metabolism.
Ultrastructural study of the mature egg of the marine sponge Stelletta grubii (porifera demospongiae)
Authors: Sciscioli Margherita , Liaci Lidia Scalera , Lepore Elene , Gherardi Miriam , Simpson Tracy L. 
Published: February 2005

AbstractStelletta grubii is an oviparous demosponge, which, during its reproductive period from summer to autumn, has small eggs (80–90 μm) dispersed uniformly in the mesohyl. The nucleolated nucleus is surrounded by dictyosomes containing small vesicles, which contribute to form reserve material. Vesicles, numerous food vacuoles, and groups of mitochondria are observed in the granular cytoplasm. Electron‐dense yolk inclusions and lipids are found peripherally. The cortical portion of the egg cytoplasm possesses vacuoles with fibrillar contents. The egg forms pseudopodia, which could permit the capture of numerous bacteria present in the surrounding mesohyl. A thick layer of collagen fibrils, including lophocytes, separates the egg from the surrounding sponge mesohyl. Ultrastructural analysis has demonstrated the presence both of cellular components capable of autosynthetic activity (nutrient vesicles) and of phagocytosis mechanisms (pseudopod capture of bacteria) for the storage of nutrients by the egg.
Association of exogenous DNA with cattle and insect spermatozoa in vitro
Authors: Atkinson Peter W. , Hines Eric R. , Beaton Sandra , Matthaei Klaus I. , Reed Ken C. , Bradley Mark P. 
Published: February 2005

AbstractSpermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina) and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.
Gamete self‐discrimination in ascidians: A role for the follicle cells
Authors: De Santis Rosaria , Pinto Maria Rosaria 
Published: February 2005

AbstractGamete self‐incompatibility in the hermaphrodite tunicate Ciona intestinalis is a useful system with which to study self‐nonself recognition. We have used in vitro fertilization of oocytes isolated from the gonad of Ciona intestinalis to identify the cellular source of self‐sterility elements present on the egg envelopes. Here we show for the first time that self‐discrimination, which occurs on the egg vitelline coat, is established there in late oogenesis and is contributed or controlled by products of the overlying follicle cells. The acquisition of self‐sterility by the oocyte is prevented by the ionophore monensin, which suggests that the follicle cell self‐sterility controlling factor is a glycoprotein.
Fertilizing capacity of epididymal and testicular spermatozoa microinjected under the zona pellucida of the mouse oocyte
Authors: Lacham Orly , Trounson Alan 
Published: February 2005

AbstractMouse spermatozoa were obtained from the testis and caput, corpus, and cauda epididymis incubated for 2 h in capacitation medium and a single spermatozoon from the capacitated samples microinjected into the pervitelline space of mature mouse oocytes. Spermatozoa from the testis were unable to fertilize oocytes and few spermatozoa from the caput were capable of fertilization (0–7% depending on the method of preparation). Similar fertilization rates (30–45%) were obtained with spermatozoa with forward progressive motility from the proximal and distal corpus and the cauda epididymis, but those that were vibratory or with local circular motility had significantly reduced fertilizing capacity (6–10%). The capacity of spermatozoa from the different regions of the testis and epididymis to bind to zona‐free oocytes followed the same pattern as fertilization rate after microinjection. There was a progressive increase in acrosome reactions after 2 h incubation in capacitation medium in samples obtained from the testis to the cauda epididymis. Maturation of the capacity to bind to the perivitelline membrane and to develop forward progressive motility, rather than the capacity to acrosome react, appears to govern the fertilization of oocytes in which the zona has been bypassed by microinjection. These characteristics are obtained in the proximal segment of the corpus. However, there was evidence that the embryo developmental capacity of oocytes fertilized by spermatozoa from the higher segments of the epididymis is reduced, particularly in oocytes fertilized by caput spermatozoa. These observations suggest that sperm microinjection may have only a limited benefit for improving fertilization rates for men with high epididymal obstructive azoospermia.