bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of oxygen toxicity on early development of mouse embryos
Authors: Umaoka Yoh , Noda Yoichi , Narimoto Katsuhiko , Mori Takahide
Published: February 2005

To examine the effects of oxygen toxicity on embryonic development, mouse pronuclear embryos were cultured under low oxygen conditions with or without superoxide dismutase (SOD), and the blastulation rate was compared with that of embryos cultured under standard conditions. The blastulation rate of mouse pronuclear embryos cultured under standard conditions was only 1.5% (2/131). This rate was increased significantly, to 28.5% (43/151), when the embryos were cultured under low oxygen conditions; and to 31.0% (35/113) when SOD (500 μg/ml) was added to the medium under standard conditions; the rate was increased to 75.2% (115/153) when the embryos were cultured under low oxygen conditions in the presence of SOD. The minimum effective concentration of SOD in the culture medium was 50 μg/ml under conditions of 5% O2. The blastulation rate was significantly decreased after 1‐hr exposure of pronuclear embryos to room atmospheric oxygen concentration (20% O2), and subsequent culture under 5% O2 with SOD did not result in an improved blastulation rate. Culture with SOD under 5% O2 promoted the development of two‐cell stage embryos to the blastocyst stage. When two‐cell stage embryos were collected 48 hr after hCG and cultured for 66 hr, their blastulation rate was similar to that of embryos collected from mice 114 hr after hCG. These results suggested that embryonic development in vitro is greatly affected by atmospheric oxygen throughout the early embryonic stages and that this harmful effect can be prevented by culturing embryos under low oxygen conditions and in the presence of SOD.
Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos
Authors: Rose Teresa A. , Bavister Barry D.
Published: February 2005

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2–5 mm follicles of ovaries collected at a local abattoir. The oocyte‐cumulus complexes (OCCs) were cultured for 23–25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 μg/ml luteinizing hormone (LH), 0.5 μg/ml follicle‐stimulating hormone (FSH), and 1 μg/ml estradiol. After maturation for 23–25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum‐free embryo culture medium (HECM‐1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEMα, TCM199, MEMα/+, RPMI:MEMα) better supported normal oocyte maturation as determined by embryo development to the two‐cell (76–82%), morula/blastocyst (25–32%), and blastocyst (12–19%) stages. Oocytes that were matured in Waymouth's medium MB 752/1 or Ham's F‐12 had a significantly reduced incidence of cleavage to the two‐cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two‐cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts. This study demonstrates that (1) the culture medium used for bovine oocyte maturation can markedly affect subsequent embryo development at several stages, including blastocysts, and (2) bovine oocytes can be matured and fertilized and develop to blastocysts in serum‐free medium.
Perivitelline space of mammalian oocytes: Extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization
Authors: Dandekar P. , Talbot P.
Published: February 2005

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.
Expression of ribosomal protein genes in mouse oocytes and early embryos
Authors: Taylor Kent D. , Pikó Lajos
Published: February 2005

The quantitative changes in the mRNAs for ribosomal proteins L7a, L18a, and S15 were assayed in slot hybridization experiments using labeled cRNA probes with total RNA from late growth‐phase oocytes, ovulated eggs, and early embryos through the blastocyst stage. All three mRNAs showed a similar developmental pattern of prevalence, but their copy numbers per oocyte or embryo fluctuated according to developmental stage. There are on an average about 17,000 copies of each mRNA in the late growth‐phase oocyte; this number drops to one‐fifth to one‐tenth in the ovulated egg and two‐cell embryo but increases rapidly during cleavage to about 25,000 in the eight‐cell embryo and about 42,000 in the blastocyst. A comparison of the levels of these mRNAs with the reported rates of ribosomal protein synthesis (LaMarca and Wassarman, 1979) suggests that, in late growth‐phase oocytes, ribosomal protein synthesis is regulated primarily at the translational level and is kept low by some factor limiting mRNA utilization. On the other hand, the high rate of ribosome biosynthesis during early embryogenesis from the two‐cell stage onward appears to involve the coordinate activation and transcription of ribosomal RNA and ribosomal protein genes coupled with the immediate translational utilization of ribosomal protein mRNAs.
Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media
Authors: Lawitts J. A. , Biggers John D.
Published: February 2005

A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCl, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage. Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium. Even in the presence of 5 mM glucose, 70% of the embryos develop to at least four cells, and 60% reach the blastocyst stage. Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development. Moreover, the response to glutamine depends on the concentration of NaCl in the medium. When the NaCl concentration is low, glutamine inhibits development. In contrast, when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt. We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte.
U2 small nuclear RNA localization and expression during bovine preimplantation development
Authors: Watson Andrew J. , Arcellana‐Panlilio Mayi , Schultz Gilbert A. , Wiemer Klaus E.
Published: February 2005

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation‐stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two‐cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (>32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one‐cell and eight‐to 16‐cell bovine embryo stages but increased 4.4‐fold between the eight‐ to 16‐cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.
Natural degenerating mitochondria in ovarian follicles of a cyprinodontidae fish, Epiplatys spilargyreus (teleost)
Authors: Thiaw Omar Thiom , Mattei Xavier
Published: February 2005

This study examines the evolution of mitochondria in the follicular cells during the development of the ovarian follicle in the teleostean fish Epiplatys spilargyreus. The mitochondria are few in number until the end of previtellogenesis; their matrix is dense, and their cristae are well developed. They proliferate during vitellogenesis and then are modified by deterioration of their matrix. Multilamellar structures are organized in the vacuolized mitochondria. During postvitellogenesis, these modifications become more advanced. The mitochondria degenerate, leaving vacuoles that contain heterogeneous structures, which will be released into the intercellular spaces. At the end of these mitochondrial transformations, the follicular cells degenerate. They release the elements which will participate in forming the secondary envelope. © 1992 Wiley‐Liss, Inc.
TGF‐β receptors
Authors: Massagué Joan , Andres Janet , Attisano Liliana , Cheifetz Sela , López‐Casillas Fernando , Ohtsuki Masahiko , Wrana Jeffrey L.
Published: February 2005

The nature and role of cell surface proteins that bind members of the TGF‐β family has been investigated. TGF‐β, activins, and BMPs each bind to receptors of 55 kDa (type I) and 70 kDa (type II). In the TGF‐β system, these receptors are implicated in the mediation of multiple responses. A member of the type II receptor family has been cloned that encodes four alternatively spliced versions of a transmembrane serine/threonine kinase receptor related to the recently cloned mouse activin receptor and C‐elegans daf‐1 gene. Inhibitors of serine/threonine kinase activity block transcriptional and growth inhibitory responses to TGF‐β. In addition to the signaling receptors, many cell types express the TGF‐β binding proteoglycan betaglycan. Betaglycan has been purified, molecularly cloned, and shown to bind TGF‐β via its core protein and basic fibroblast growth factor via its heparan sulfate chains. In addition to receptors I and II and betaglycan, some cells express a newly identified set of membrane proteins that specifically bind either TGF‐β1 or TGF‐β2. Three of the four isoform‐restricted binding proteins are bound to the membrane via phospholipid anchors. Like betaglycan, these proteins might function to regulate the interaction between TGF‐β and their target cells. © 1992 Wiley‐Liss, Inc.
TGF‐β family factors in Drosophila morphogenesis
Authors: Hoffmann F. Michael
Published: February 2005

Many Drosophila genes have now been identified with substantial sequence similarity to vertebrate protooncogenes and growth factors. Some of these have been isolated directly by cross‐hybridization with vertebrate probes and some have been recognized in the sequences of genes cloned because of their intiguing mutant phenotypes. An example of a gene isolated for its interesting development functions but with homology to a vertebrate growth factor is the Drosophila decapentaplegic gene (dpp). An example of a Drosophila gene isolated by virtue of its sequence conservation is the vgr/60A gene. Both dpp and vgr/60A are members of the transforming growth factor‐β family and are most similar to the human bone morphogenetic proteins. The regulation of the dpp gene by several different groups of pattern formation genes including the dorsal/ventral group, the terminal group, the segment polarity genes, and the homeotic genes indicates that many events in embryogenesis require the cell to cell communication mediated by the secreted dpp protein. The temporal and spatial pattern of vgr/60A expression differs from that of dpp indicating that it may be regulated by different pattern information genes. The experimental advantages of the Drosophila system should permit a better understanding of the importance of growth factor homologs in specific developmental events, aid in establishing the functional interactions between these regulatory molecules, and identify new genes that are important for the biological functions of growth factors. It is likely that some of the newly identified genes will have vertebrate homologs and the analysis of these may be helpful in studies on vertebrate development and tumor biology. © 1992 Wiley‐Liss, Inc.
The sperm entry site during fertilization of the zebrafish egg: Localization of actin
Authors: Hart Nathan H. , Becker Karen A. , Wolenski Joseph S.
Published: February 2005

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5‐ to 6‐nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15–20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.
Rabbit blastocysts accumulate platelet‐activating factor (PAF) and lyso‐PAF in vitro
Authors: Jones M. A. , Kudolo G. B. , Harper M. J. K.
Published: February 2005

Platelet‐activating factor (1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphorylcholine; PAF) is a very potent phospholipid, which has been demonstrated to stimulate smooth muscle and change vascular permeability. PAF has been detected in the rabbit preimplantation uterine endometrium and has been demonstrated to bind specifically to rabbit uterine membranes. To evaluate the possible role of PAF in maternal‐embryonic chemical communication, we report here that rabbit blastocysts can accumulate [3H]PAF from their environment. Blastocysts were able to accumulate [3H]PAF as time‐, buffer‐, age‐, and concentration‐dependent functions. The accumulation was inhibited by some PAF receptor antagonists, such as U66985, as well as by unlabeled PAF and lyso‐PAF, indicating that the accumulation process may be receptor mediated. The data support the current model of PAF as a paracrine factor in preimplantation stages of reproduction.
Purification and characterization of a vitelline coat lysin from Ciona intestinalis spermatozoa
Authors: Marino Rita , De Santis Rosaria , Hirohashi Noritaka , Hoshi Motonori , Pinto Maria Rosaria , Usui Noriko
Published: February 2005

In Ciona intestinalis a chymotrypsin‐like activity is involved in sperm penetration of the egg vitelline coat. A chymotrypsin‐like enzyme has been purified from spermatozoa by a protocol including ion exchange chromatography, gel filtration, and native polyacrylamide gel electrophoresis. The purified enzyme resulted homogeneous when analyzed by SDS‐PAGE. The molecular weight of the chmotrypsin‐like enzyme was estimated to be 35 KDa by gel filtration and 24 KDa by SDS‐PAGE in nonreducing conditions. The pH optimum of the enzyme is 8.4 and its activity is enhanced by Ca2+. It shows the highest activity towards the synthetic substrate Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Furthermore, by electron microscopy, the purified enzyme affects the structure of egg vitelline coat, and thus it fulfills one of the criteria of a lysin.
Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor
Authors: Hendrix E. M. , Mao S. J. T. , Everson W. , Larsen W. J.
Published: February 2005

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme‐linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site‐specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43‐positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface. © 1992 Wiley‐Liss, Inc.
F‐actin in guinea pig spermatozoa: Its role in calmodulin translocation during acrosome reaction
Authors: Moreno‐Fierros Leticia , Hernández Enrique Othon , Salgado Zaira O. , Mújica Adela
Published: February 2005

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F‐actin and a possible function for it been presented. In this work, actin distribution and F‐actin were determined in uncapacitated, capacitated, and acrosomal‐reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine‐phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS‐PAGE and Western blots immunostained with monoclonal and polyclonal anti‐actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll‐purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F‐actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility. © 1992 Wiley‐Liss, Inc.
A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa
Authors: Tateno Hiroyuki , Kamiguchi Yujiroh , Mikamo Kazuya
Published: February 2005

Superovulated hamster oocytes were cryopreserved and thawed according to our carefully designed procedures. More than 90% (92 ± 4%) of oocytes survived freezing and thawing. They were proven to be well conserved, showing excellent performance comparable to freshly ovulated oocytes in the human sperm penetration test (proportion of penetrated ova: 94.7% vs. 93.6%) and human sperm chromosome analysis (proportion of metaphasic ova: 81.8% vs. 83.6%). There was no statistically significant difference in the incidences of sperm chromosome aberrations between assays using fresh and frozen‐thawed oocytes. In addition, there was no statistically significant increase of aberrations in female pronuclear (hamster) chromosomes. This freezing‐thawing method was found to be reliable, yielding viable hamster oocytes of high quality. © 1992 Wiley‐Liss, Inc.
In vitro fertilization, development, and implantation after exposure of mature mouse oocytes to visible light
Authors: Barlow P. , Puissant F. , Van Der Zwalmen P. , Vandromme J. , Trigaux P. , Leroy F.
Published: February 2005

Mature mouse oocytes were exposed prior to in vitro fertilization to visible light during 1, 2, or 4 hr at an intensity of 4,000 lux. Compared to controls cultured under identical conditions but protected from light, exposed eggs did not show any significant modification of cleavage speed are rate. After transfer of blastocysts obtained in vitro in uteri of pseudopregnant females, the implantation rate and the proportion of normal fetuses were not found to be different in relation to preliminary light exposure of oocytes fertilized and cultured in vitro. © 1992 Wiley‐Liss, Inc.
Determination of enzyme activities of energy metabolism in the maturing rat oocyte
Authors: Tsutsumi Osamu , Satoh Kazuo , Taketani Yuji , Kato Takahiko
Published: February 2005

Enzyme activities were determined quantitatively in individual rat oocytes to study their energy metabolism during maturation. Low hexokinase activity and high activities of lactate dehydrogenase and enzymes in the phosphate pathway, i.e., glucose 6‐P and 6‐P gluconate dehydrogenases, were characteristic of immature oocytes. Hexokinase may be a rate‐limiting enzyme that enables oocytes to use glucose as an energy source. During maturation, the activities of hexokinase, phospho‐fructokinase, and malate dehydrogenase increased significantly, suggesting that the glycolytic pathway, as well as the tricarboxylic acid cycle, developed as the first meiotic division proceeded. In contrast, the activities of glucose 6‐P and 6‐P gluconate dehydrogenases decreased in maturing oocytes. The observation that the enzyme pattern in mature oocytes resembles more closely that in somatic cells appears to be significant, especially in light of previous studies showing this developmental trend in preimplantation embryos. © 1992 Wiley‐Liss, Inc.
Isolation and cultivation of blastocyst‐derived stem cell lines from american mink (Mustela vison)
Authors: Sukoyan M. A. , Golubitsa A. N. , Zhelezova A. I. , Shilov A. G. , Vatolin S. Y. , Maximovsky L. P. , Andreeva L. E. , McWhir J. , Pack S. D. , Bayborodin S. I. , Kerkis A. Y. , Kizilova H. I. , Serov O. L.
Published: February 2005

Ten embryonic stem (ES) cell lines from mink blastocysts were isolated and characterized. All the lines had a normal diploid karyotype; of the ten lines studied, five had the XX and five had the XY constitution. Testing of the pluripotency of the ES‐like cells demonstrated that (1) among four lines of genotype XX, an X was late‐replicating in three; both Xs were active in about one‐third of cells of line MES8, and analysis of glucose‐6‐phosphate dehydrogenase revealed no dosage compensation for the X‐linked gene; (2) when cultured in suspension, the majority of lines were capable of forming “simple” embryoid bodies (EB), and two only showed the capacity for forming “cystic” multilayer EBs. However, formation of ectoderm or foci of yolk sac hematopoiesis, a feature of mouse ES ceils, was not observed in the “cystic” EB; (3) when cultured as a monolayer without feeder, the ES cells differentiated into either vimentin‐positive fibroblast‐like cells or cytokeratin‐positive epithelial‐like cells (less frequently); neural cells appeared in two lines; (4) when injected into athymic mice, only one of the four tested lines gave rise to tumors. These were fibrosarcomas composed of fibrobalst‐like cells, with an admixture of smooth muscular elements and stray islets of epithelial tissue; (5) when the ES cells of line MES1 were injected into 102 blastocyst cavities and subsequently transplanted into foster mathers, we obtained 30 offspring. Analysis of the biochemical markers and coat color did not demonstrate the presence of chimaeras among offspring. Thus the cell lines derived from mink blastocysts are true ES cells. However, their pluripotential capacities are restricted. © 1992 Wiley‐Liss, Inc.
Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes
Authors: Collas Philippe , Fissore Rafael , Robl James M. , Sullivan Eddie J. , Barnes Frank L.
Published: February 2005

The influence of electrical stimulation on the level of intracellular Ca2+ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca2+ concentration was determined with the Ca2+ indicator fura‐2 dextran (fura‐2 D). The Ca2+ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca2+ rise from baseline (≈︁ 12 nM), a short‐duration Ca2+ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long‐duration Ca2+ rise (from 12 nM to 1,000–2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca2+ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca2+ rise, and at 1.0 kVcm−1 for 40 μsec, all oocytes displayed a long‐duration Ca2+ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca2+ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca2+ rises. This mannitol‐induced Ca2+ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca2+ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca2+ stimulation impaired development. Optimum development was obtained with (1) three pulses of 0.2 kVcm−1 for 20 μsec, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or (2) three pulses of 1.0 kVcm−1 for 20 μsec after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca2+ rise prior to the pulse. The results indicate that the level of Ca2+ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca2+ response, activation, and parthenogenetic development. © 1993 Wiley‐Liss, Inc.
In vitro maturation of oocytes from a marsupial, the tammar wallaby (Macropus eugenii)
Authors: Mate Karen E. , Rodger John C.
Published: February 2005

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO2 in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell‐intact or granulosa cell‐free or in the presence of 10 IU ml−1 PMSG or 10 μg ml−1 porcine luteinizing hormone (LH) + 10 μg ml−1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5‐mm‐diameter) and large (≥1.5‐mm‐diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA‐specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley‐Liss, Inc.
Gene introduction into mouse blastocysts via “pricking”
Authors: Sato Masahiro , Tada Norihiro , Iwase Reiko , Amann Egon
Published: February 2005

It is a well‐known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation ‘pricking’ technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid‐gestation stage were obtained. Twenty‐two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)‐Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli β‐galactosidase (β‐gal) gene to a mouse metallothionein‐I (MT‐I) promoter and assessed for β‐gal activity histochemically after 1 and 5 days of culture in the presence of 1 μM CdCI2, at least 65% of the embryos exhibited β‐gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique. © 1993 Wiley‐Liss, Inc.
The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro
Authors: Biggers J. D. , Lawitts J. A. , Lechene C. P.
Published: February 2005

The development of outbred mouse (CF1) zygotes in vitro has been studied using medium SOM in which the concentrations of NaCl (85, 105, 125 mM), glutamine (0, 1, 2 mM), and betaine (0, 1, 2 mM) were varied. The effects of the compounds were studied using a 33 factorial experimental arrangement. The inhibitory effect of relatively high concentrations of NaCl and the protective effect of glutamine were confirmed. Betaine, an organic osmolyte, can also protect against the deleterious effects of relatively high concentrations of NaCl. The intracellular contents of potassium and sodium have also been measured in single zygotes using X‐ray electron probe spectrometry. When medium SOM contains 85 mM or 125 mM NaCl, the intracellular content of Na rises and the content of K decreases. These changes are partially reduced in the presence of 125 mM NaCl if betaine is also in the medium. Betaine has no effect on the intracellular content of K and Na if the concentration of NaCl is 85 mM. These results suggest that organic osmolytes may be required in embryo culture media to prevent excessive changes in the intracellular ionic concentration. © 1993 Wiley‐Liss, Inc.
Members of the rafgene family exhibit segment‐specific patterns of expression in mouse epididymis
Authors: Winer M. A. , Wadewitz A. G. , Wolgemuth D. J.
Published: February 2005

The proto‐oncogene c‐raf‐1 and the related genes A‐raf and B‐raf encode serine/threonine protein kinases thought to be involved in regulating gene expression by acting as part of second‐messenger signaling pathways within the cell. Among the tissues in which A‐raf and c‐raf‐1 have been shown to be expressed was mouse epididymis. The present studies were undertaken to determine if the raf family genes exhibited specificity in their pattern of expression that might be indicative of specific function in the epididymis. Northern and in situ hybridization analyses demonstrated that c‐raf‐1 mRNA was expressed as a 3.1 kb transcript at uniform levels throughout the length of the epididymis in all types of epididymal epithelial cells. Neither the germ cell‐specific testicular transcripts nor the somatic transcripts of B‐raf were detected by either Northern or in situ hybridization analysis in any region of the epididymis. A‐raf, expressed as two transcripts of 2.6 and 4.3 kb, was the only gene examined which exhibited a segment‐specific pattern of expression, being highest in the principal epithelial cells of the proximal caput epididymis and decreasing progressively in more distal regions of the tubule. These studies indicate that each raf gene exhibits a characteristic pattern of expression in the epididymis; A‐raf in particular may play a unique regulatory role in the regionalized functions of the epididymis. © 1993 Wiley‐Liss, Inc.
Gene expression, X‐inactivation, and methylation during spermatogenesis: The case of Zfa, Zfx, and Zfy in mice
Authors: Erickson Robert P. , Zwingman Theresa , Ao Asangla
Published: February 2005

While it has become clear that X‐inactivation in the female soma is complete in mouse (in contrast to being “patchy” in man), the degree of X‐inactivation in the testes has not been ascertained. We have compared autosomal and X‐linked zinc finger homolog expression and X‐linked and Y‐linked zinc finger homolog methylation in an attempt to elucidate this question. Using RTPCR, we have extended earlier studies of Zfx and Zfa expression in developing testes and find that Zfa expression starts at the time of X‐inactivation while Zfx expression is continuous. Cell separation studies did not preclude continued expression of Zfx in adult germ cells. The methylation status of four CCGG residues in the Zfx promoter was studied using PCR bridging this region before and after DNA digestion with the isoschizomers Msp I and Hpa II, the latter being methylation sensitive. Hpa II resistant Zfx promoter DNA was found in all female tissues, but not in male tissues, including the testes. Previous studies have shown that Zfy is expressed at meiosis (like Zfa and unlike Zfx). Despite its expression, the Zfy gene is adjacent to, or contains, highly methylated CCGG sites since hybridization after Msp I digestion detected multiple small fragments that were not released after DNA digestion with Hpa II. Thus, Zfx is not methylated in sperm, while Zfy is, in contrast to their apparent patterns of expression. © 1993 Wiley‐Liss, Inc.
Spermatid nucleus of Megaselia scalaris loew (insecta, diptera, phoridae): A study using anti‐histone antibodies, scanning electron microscopy, and a centromere‐specific oligonucleotide
Authors: Wolf Klaus Werner , Jeppesen Peter , Mitchell Arthur
Published: February 2005

The structure of elongated spermatid nuclei was examined in the fly Megaselia scalaris using indirect immunofluorescence with anti‐histone antibodies, scanning electron microscopy, and in situ hybridization with a centromere‐specific oligonucleotide. The immunofluorescence experiments showed that, in keeping with the situation in most animals, histones are hyperacetylated prior to their displacement from the nucleus in the course of spermiogenesis. Scanning electron microscopy revealed that grooves run parallel to the long axis of the spermatid nuclei. The chromatin is segmented into blocks lateral to the grooves. This finding most probably indicates that the chromatin is not yet maximally condensed at this stage. The retarded chromatin condensation may be correlated with the export of somatic histones from the nucleus. The location of the centromeres could not be identified using scanning electron microscopy, but in situ hybridization showed that a centromere‐specific oligonucleotide mapped to the central or close to the central areas within the spermatid nucleus. Possibly, the chromosomes are extended and arranged parallel to the long axis of the spermatid nucleus. © 1993 Wiley‐Liss, Inc.
Multifactorial regulation of IGF‐I gene expression
Authors: Rotwein Peter , Bichell David P. , Kikuchi Kiyoshi
Published: February 2005

Insulin‐like growth factor‐I (IGF‐I) is a highly conserved 70‐residue circulating peptide with diverse biological effects. In mammals IGF‐I is an essential mediator of normal postnatal growth and its expression is influenced by hormonal, nutritional, tissue‐specific, and developmental factors. Recent studies have demonstrated that the IGF‐I gene is more complicated than might have been predicted from its simple protein sequence. In rats and in humans the single‐copy six‐exon gene is transcribed by adjacent promoters into nascent RNAs with different 5′ leader sequences that undergo both alternative RNA splicing and differential polyadenylation to yield multiple mature transcripts. These observations suggest that trophic agents may modulate expression of IGF‐I at any of several nodal points. In this report we review several of the mechanisms responsible for regulating production of IGF‐I in the rat. During neonatal development IGF‐I gene transcription is progressively activated leading to a rise in both hepatic IGF‐I mRNA and in serum IGF‐I. The induction of IGF‐I expression is limited to mRNAs directed by promoter 1, the more 5′ of two rat IGF‐I gene promoters, and precedes the ontogenic appearance of liver growth hormone (GH) receptors, indicating that mechanisms independent of GH activate IGF‐I expression during early postnatal life. By contrast, in adult GH‐deficient rats, a single intraperitoneal injection of GH causes a prompt rise in IGF‐I gene transcription that is mediated equivalently by promoters 1 and 2. Transcriptional induction occurs within 30 min of GH treatment and is associated with a transiently appearing DNase I hypersensitive site in the second IGF‐I intron. These two physiological models show that IGF‐I expression is mediated by at least two distinct transcriptional mechanisms. A challenge for the future will be to define the transcription factors and delineate the critical steps in the regulation of a growth factor that is essential for normal growth and maturation. © 1993 Wiley‐Liss, Inc.
Developmental expression of the IGF‐II/mannose 6‐phosphate receptor
Authors: Nissley Peter , Kiess Wieland , Sklar Mark
Published: February 2005

The first indication that the insulin‐like growth factor‐II/mannose 6‐phosphate receptor (IGF‐II/M6PR) is developmentally regulated came from studies of the serum form of the receptor in the rat. By immunoblotting, the circulating form of the receptor, which was 10 kDa smaller than the tissue receptor, was high in 19 day fetal and 3, 10, and 20 day postnatal sera and then declined sharply. We next used quantitative immunoblotting to measure the total tissue IGF‐II/M6PR in the rat. The receptor levels were high in fetal tissues and in most tissues declined dramatically in late gestation and/or in the early postnatal period. The rank order of receptor expression was heart > placenta > lung = intestine > muscle = kidney > liver > brain. In heart, the receptor was 1.7% of total protein in the extract. More recently, we have examined the expression of IGF‐II/M6PR mRNA using Northern blotting and a solution hybridization/RNase protection assay. The rank order of receptor mRNA concentration among fetal tissues agreed with the rank order of receptor protein. The concentration of receptor mRNA was significantly lower in postnatal tissue than in fetal tissue. Thus IGF‐II/M6PR mRNA concentration is an important determinant of receptor protein in most tissues. What is the function of the IGF‐II/M6PR in embryonic and fetal tissues? The M6PR in birds and frogs does not bind IGF‐II. It is intriguing that the rat IGF‐II/M6PR is prominent during the embryonic and fetal periods, times at which the differences between mammals, on the one hand, and frogs and birds, on the other, are most striking. Tissue remodeling is an important feature of embryonic and fetal development. Therefore, the well‐established lysosomal enzyme targeting function of the receptor may be of particular importance. Since IGF‐II can inhibit the cellular uptake of lysosomal enzymes via the IGF‐II/M6PR, IGF‐II may modulate this lysosomal enzyme targeting function. In addition, the receptor can provide a degradative pathway for IGF‐II by receptor‐mediated internalization. Thus the receptor could provide a check on the high levels of IGF‐II known to be present in the fetus. Finally, the IGF‐II/M6PR could directly signal certain biologic responses to IGF‐II. © 1993 Wiley‐Liss, Inc.
Expression of bovine trophoblast interferons by in vitro‐derived blastocysts is correlated with their morphological quality and stage of development
Authors: Hernandez‐Ledezma José Juan , Mathialagan Nagappan , Villanueva Cesar , Sikes John D. , Roberts R. Michael
Published: February 2005

Bovine embryos, whether produced naturally or by in vitro techniques, exhibit considerable variability in morphological quality and develop at different rates. Our objectives have been to determine whether initial expression of trophoblast interferon (IFN‐τ) was a reflection of conceptus stage of development or age and whether there was an effect of embryo quality on the amount of IFN‐τ produced. Early blastocysts (N =187) were selected at the onset of blastocoele formation and cultured individually. Embryo quality (excellent, good, or fair: E, G, or F) and developmental stage (early, expanded and hatched blastocysts: BL, EBL, and HBL, respectively) were used in a 3 x 3 factorial complete randomized block design, each block (n = 4) consisting of batches of embryos produced from oocytes in different collections. Quality and developmental stage of embryos and IFN‐τ released into the medium were assessed every 24 h. Production of IFN‐τ (units/embryo/24 h) was greater (P < 0.01) among hatched blastocysts (HBL; 0.91 ± 0.08) than expanded blastocysts (EBL; 0.23 ± 0.04) and early blastocysts (BL; 0.05 ± 0.08). Embryos of similar developmental stage but differing by 2 days in age released equal amounts of IFN‐τ. Expression of antiviral activity increased (P < 0.05) from 27% to 57% to 100% as development proceeded from BL to EBL and to HBL respectively. More IFN‐τ was produced by HBL graded G (1.0 ± 0.1) or E (1.3 ± 0.1) than by those of F quality (0.5 ± 0.1). All blastocysts, whatever their quality and developmental stage, contained IFN‐± mRNA. Therefore, developmental stage and quality of the embryos significantly influence the expression of IFN‐±. The amount produced may be a useful objective indicator of embryo quality. © 1993 Wiley‐Liss, Inc.
Chromatin and microtubule morphology during the first cell cycle in bovine zygotes
Authors: Long Charles R. , Pinto‐Correia Clara , Duby Robert T. , De Leon F. Abel Ponce , Boland Maurice P. , Roche James F. , Robl James M.
Published: February 2005

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage. © 1993 Wiley‐Liss, Inc.