bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin family growth factors have specific effects on protein synthesis in preimplantation mouse embryos
Authors: Shi C. Z. , Collins H. W. , Buettger C. W. , Garside W. T. , Matschinsky F. M. , Heyner S.
Published: February 2005

Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight‐cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF‐I, and IGF‐II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35–50 embryos into human tubal fluid (HTF) medium containing L‐[35S]‐methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2‐D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer‐assisted analysis, we were able to identify proteins that showed a statistically significant (P < 0.05) change in synthesis. At the eight‐cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin‐treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF‐I at the eight‐cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight‐cell stage embryos incubated with IGF‐II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis. However, seven IGF response proteins were found to be proteins that showed significant changes in isotope incorporation during the eight‐cell to blastocyst stage of development (Shi et al., 1993). In all, 54 proteins were affected, and these were unique; thus, protein synthesis in preimplantation mouse embryos is influenced by insulin and the IGFs, and further, each growth factor affects specific proteins. © 1994 Wiley‐Liss, Inc.
One‐minute exposure of 4‐cell mouse embryos to glucose overcomes morula block in CZB medium
Authors: Chatot Clare L. , Lewis‐Williams Jennifer , Torres Ioannis , Ziomek Carol A.
Published: February 2005

One‐cell mouse embryos that block at the 2‐cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1‐min exposure to 27 mM glucose at the 4‐cell stage (∼42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67–73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts). © 1994 Wiley‐Liss, Inc.
Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture
Authors: Nagai T. , Takenaka A. , Mori T. , Hirayama M.
Published: February 2005

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca2+. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine‐containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs‐free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca2+ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca2+ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca2+ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca2+ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley‐Liss, Inc.
Redistribution of nuclear antigens linked to cell proliferation and RNA processing in mouse oocytes and early embryos
Authors: Vautier Dominique , Besombes Didier , Chassoux Danielle , Aubry Florence , Debey Pascale
Published: February 2005

We have systematically analyzed by indirect immunofluorescence the subcellular distribution of nuclear antigens in relation to developmental stages of maturing mouse oocytes and developing embryos. Antigens were of two types: (1) a protein whose nuclear localization in interphase somatic cells depends on their proliferative state protein recognized by a monoclonal antibody 43B1N, and (2) snRNP polypeptides recognized by autoimmune sera of anti‐Sm and anti‐RNP type. The protein recognized by 43B1N was present in the germinal vesicle of oocytes from antral follicles, but absent from the nuclei during the first hours of embryonic life up to the middle to late 2‐cell stage. Starting from this stage, it was always found in nuclei of interphase blastomeres, where its “speckles”; co‐localized with the speckles containing high concentrations of snRNP polypeptides. SnRNP polypeptides recognized by anti‐Sm and anti‐RNP sera were in turn found in nuclei of all developmental stages. When embryos were treated with aphidicolin or cytochalasin D to arrest cell division, the 43B1N reacting protein was again localized in the pronuclei at 42 hr post‐hCG, i.e., slightly later than the onset of transcriptional activity. These results suggest a progressive building up of nuclei during embryonic development, which could influence gene expression. © 1994 Wiley‐Liss, Inc.
Temporal and spatial expression of leukemia inhibitory factor in rabbit uterus during early pregnancy
Authors: Yang Zeng‐Ming , Le Su‐Ping , Chen Dong‐Bao , Harper Michael J. K.
Published: February 2005

Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to document the appearance of LIF in the rabbit uterus during early pregnancy and to determine whether changes just prior to implantation, similar to those in mice, occurred. LIF was localized in endometrial epithelium, myometrium, and endometrial glands. A low level of LIF was detected in the uterus of nonestrous and estrous females. LIF expression reached its highest level on day 5 of pregnancy and declined on days 6 and 7. By day 13 of pregnancy, little endometrial LIF was apparent. The expression of LIF on day 5 of pseudopregnancy was similar to that on day 5 of pregnancy. LIF expression was much higher at implantation sites than that at nonimplantation areas on day 7 of pregnancy. It is concluded that LIF may be important for the implantation of rabbit blastocysts. © 1994 Wiley‐Liss, Inc.
Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation
Authors: Tatone Carla , Van Eekelen Christina Grietje , Colonna Rosella
Published: February 2005

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona‐free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1‐oleoyl‐2‐acetyl‐sn‐glycerol) and inseminated after different periods. Eggs challenged with sperm at 30‐ or 60‐min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120‐min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 μM external Ca2+ or to heat shock), a normal ability to be penetrated was recorded at 30‐min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5‐trisphosphate into the egg at 1 μM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur. © 1994 Wiley‐Liss, Inc.
Lipovitellin 2β is the 31 kD Ni2+‐binding protein (pNiXb) in Xenopus oocytes and embryos
Authors: Grbac‐Ivankovic Svetlana , Antonijczuk Katarzyna , Varghese Alison H. , Plowman Marilyn C. , Antonijczuk Adam , Korza George , Ozols Juris , Sunderman F. William
Published: February 2005

An Ni2+‐binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N‐terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2β, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2β agree in N‐terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metalblot competition assay, the abilities of metal ions to compete with 63Ni2+ for binding to pNiXb were ranked: Zn2+ ≈ Cu2+ ≈ Co2+ > Cd2+ ≈ Mn2+ > Sn2+. This study shows that Xenopus lipovitellin 2β is a metal‐binding protein in vitro, and raises the possibility that it may function similarly in vivo. © 1994 Wiley‐Liss, Inc.
Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei
Authors: Collas Philippe , Barnes Frank L.
Published: February 2005

The developmental potential of bovine inner cell mass (ICM) and somatic differentiated (granulosa cell) nuclei was investigated using nuclear transplantation. ICM blastomeres were isolated after immunosurgery of day 7 in vitro produced blastocysts and cumulus granulosa cells recovered from in vitro matured oocytes. Nuclear transplantation was carried out by microinjection of the lysed donor cells into enucleated mature oocytes. Oocytes were activated by three 0.2 kVcm−1/20 μs pulses in mannitol containing 100 μM Ca2+, with each pulse 22 min apart. Embryos were cultured in vitro for 7 days and blastocysts were transferred into recipients. ICM and granulosa cell donor nuclei directed 7% (20/304) and 9% (19/213) development to blastocysts, respectively. Fifteen blastocysts from ICM donors resulted in four pregnancies (27%) and two births. No pregnancy was detected with granulosa cell donors. The results illustrate the totipotency of ICM nuclei and indicate that granulosa cell nuclei promote preimplantation development of nuclear transplant embryos. © 1994 Wiley‐Liss, Inc.
Inhibition of flagellar motility of fowl spermatozoa by L‐carnitine: Its relationship with respiration and phosphorylation of axonemal proteins
Authors: Ashizawa Koji , Kamiya Tetsushi , Tamura Ikue , Tsuzuki Yasuhiro
Published: February 2005

The action of carnitine in regulating fowl sperm motility was investigated. As the concentration of L‐carnitine was increased (0–20 mM), the motility of intact and demembranated fowl spermatozoa was reduced at 30°C. Even the presence of 1 mM CaCl2 before the addition of 10 mM carnitine could not prevent the inhibition of motility at 30°C and 40°C. However, motility was restored by reducing the concentrations of carnitine. Carnitine also inhibited the oxygen consumption and ATP concentrations of intact spermatozoa, and caused a reduction in intracellular free Ca2+ concentrations. Phosphorylation of a 50 kDa protein and dephosphorylation of 24 kDa and 30 kDa proteins of demembranated spermatozoa were observed after the addition of carnitine. In contrast, the flagellar ATPase activity of crude dynein extract was not affected by the addition of carnitine. These results suggest that inhibitory effect of carnitine for motility may be directly on the axonemal phosphoproteins, but not directly on the dynein ATPase activity. The physiological role of carnitine for fowl spermatozoa in the ductus deferens is discussed. © 1994 Wiley‐Liss, Inc.
Expression of the complement regulatory protein CD59 on human spermatozoa: Characterization and role in gametic interaction
Authors: Fenichel P. , Cervoni F. , Hofmann P. , Deckert M. , Emiliozzi C. , Hsi B. L. , Rossi B.
Published: February 2005

Protectin (CD59) is a complement regulatory protein which blocks the membrane attack complex during complement activation. CD59 was identifield on the human sperm surface by means of H19, an IgG1 anti‐protectin mouse monoclonal antibody. Using Indirect immunofluorescence, flow cytometry and immunoperoxidase, CD59 was found to be present on the whole plasma membrane including the head and tail of fresh ejaculated, capacitated and acrosome‐reacted spermatozoa. Immunoperoxidase staining of normal testicular sections indicated that this protein was already present on intraluminal germ cells. Analysis of this sperm protein by gel electrophoresis and immunoblotting revealed that its molecular weight of 20 kDa was comparable to that of CD59 expressed on peripheral blood cells (erythrocytes, lymphocytes) and that it was bound to the membrane through a glycophospholipid tail which could be released after treatment with phosphatidylinositol‐specific phospholipase C. Associated to membrane cofactor protein (CD46) and decay accelerating factor (CD55) located in the acrosomal membranes, CD59 may participate to the protection of male gametes against complement‐mediated damage as they travel through the female genital tract. Moreover CD59, known as an adhesion molecule involved in lymphocyte rosettes, may also participate in cell to cell adhesion during gametic interaction since H19 inhibited sperm binding and reduced the penetration rate and index during the hamster egg penetration test. © 1994 Wiley‐Liss, Inc.
Pregnancy‐associated endometrial expression of antileukoproteinase gene is correlated with epitheliochorial placentation
Authors: Badinga Lokenga , Michel Frank J. , Fields Michael J. , Sharp Dan C. , Simmen Rosalia C. M.
Published: February 2005

Uterine expression of the mRNA encoding antileukoproteinase (ALP) is highest in pig uterus during mid‐ to late pregnancy, suggesting a stage of pregnancy‐dependent role for this elastase/cathepsin G protease inhibitor in feto‐maternal interactions. To examine a potential relationship between uterine synthesis of ALP and the type of placentation in mammalian species, the expression of ALP mRNA and/or protein in pregnant mares, cows, rats, and mice was evaluated. Genomic DNA and mRNA hybridization analyses were performed using a porcine ALP cDNA as probe. The concentration of ALP protein in reproductive tissues was determined by RIA using a polyclonal antibody raised against a synthetic peoptide (ALP 16P) corresponding to amino acid residues 21–36 of the porcine ALP protein. A single ALP mRNA transcript of approximately 0.8 kb in length was detected in equine and bovine uterine tissues. The relative abundance of ALP mRNA in equine endometrium increased between days 125–170 (mid‐pregnancy), and then decreased by day 215 of pregnancy. Similarly, the steady state levels of ALP mRNA in bovine endometrium and myometrium were higher during mid‐ to late than during early pregnancy. The levels of ALP mRNA in bovine fetal cotyledon were low and did not change significantly with stage of pregnancy. No hybridization was detected to pregnant rat endometrial tissues, although high stringency Southern blot analysis of porcine, bovine, and rat genomic DNAs using porcine ALP cDNA as probe predicted a high degree of nucleotide sequence homology in their respective ALP genes. In pregnant cows, concentrations of ALP protein were higher in maternal endometrium and myometrium than in fetal cotyledon. Tissue ALP content in bovine uterus increased between days 17–89, and then decreased by day 248 of pregnancy. In contrast, no ALP protein was detected in cytosolic extracts prepared from endometrium of pregnant rats and mice. THe demonstrated synthesis of ALP mRNA and/or protein in the endometrium of the mare and the cow similar to that of the pig, but not in the endometrium of the rat and mouse, during pregnancy indicates a potential correlation between endometrial ALP expression and epitheliochorial type of placentation in mammalian species. © 1994 Wiley‐Liss, Inc.
Characterization and properties of steroid binding protein in Bufo arenarum serum
Authors: Fernández Silvia N. , Mansilla‐Whitacre Zulema C. , Miceli Dora C.
Published: February 2005

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 α‐dihydrotestosterone (DHT), testosterone (T), and estradiol‐17β (E2) with high affinity (107 M−1 – 108 M−1) and fair capacity (10−6 M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady‐state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control. © 1994 Wiley‐Liss, Inc.
Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide
Authors: Presicce Giorgio A. , Yang Xiangzhong
Published: February 2005

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 μg/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2–8 cells following culture for 3 days was similar among treated groups (32–41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre‐ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One‐ to 5‐min groups resulted in 42–44% cleavage contrasted to 1–12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 μg/ml). Cleavage to 2–8 cells was similar among the 5 and 10 μg/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 μg/ml group (24%) and the controls (2–13%). When 5 μg/ml cycloheximide was used (Experiment 4), pre‐exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38–41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment. Oocytes developed to blastocyts were 0%, 14%, 3%, and 3% (P < 0.05), respectively, for control, treatment with ethanol and cycloheximide in the presence, or absence of cytochalasin B, or electrical pulse plus cycloheximide. In conclusion, the combined ethanol and cycloheximide treatment supported high rates of parthenogenetic development using 24 hr IVM bovine oocytes. Blastocyst rate was significantly higher when cytochalasin B was added to the combined activation regimen. © 1994 Wiley‐Liss, Inc.
Potential mechanisms regulating the extracellular activities of basic fibroblast growth factor (FGF‐2)
Authors: Baird Andrew
Published: February 2005

Basic fibroblast growth factor (FGF‐2) is nearly ubiquitous in its distribution, leading numerous investigators to propose that there must exist highly specific mechanisms to regulate its bioavailability. Moreover, in each of the tissues where it can be localized, numerous cells are its potential target. The identification of these mechanisms could serve as an important step in developing novel strategies to inhibit FGF action. It could be possible to block such FGF‐dependent activities as angiogenesis, tumor growth, reproduction, and selected diseases of cell proliferation. Over the course of the past several years, we have attempted to describe some of the processes that might regulate a target cell's ability to activate FGF‐2 in its local milieu. © 1994 Wiley‐Liss, Inc.
Structure‐function studies of FGF‐1: Dissociation and partial reconstitution of certain of its biological activities
Authors: Burgess Wilson H. , Friesel Robert , Winkles Jeffrey A.
Published: February 2005

We reported previously that the mitogenic activities of FGF‐1 (acidic FGF) could be dissociated from its receptor‐binding activities by site‐directed mutagenesis of lysine 132 to a glutamic acid. Although the mutant FGF‐1 protein binds to the high‐affinity tyrosinekinase receptors, stimulates tyrosine‐kinase activity, and promotes expression of immediate‐early genes, it is not mitogenic for a variety of tested cell lines. Interestingly, the mutant FGF‐1 is capable of other functions associated with the wild‐type protein such as promotion of mesoderm formation in Xenopus animal caps. The mutant exhibits a reduced apparent affinity for heparin‐Sepharose compared to the wild‐type protein. The relationship between the reduced heparin affinity and lack of mitogenic activity of this mutant is not clear. Recent data indicates the relationship is not as simple as reduced stability of the protein. When NIH 3T3 cells are transfected with expression vectors encoding either wild‐type or mutant FGF‐1, a transformed phenotype can be seen in cells overexpressing the wild‐type FGF‐1, whereas cells overexpressing mutant FGF‐1 appear normal. Analysis of lysates of these cells indicates that a tyrosine‐kinase cascade, distinct from that associated with the high‐affinity cell surface receptors, has been activated in the wild‐type transfected cells but not in the mutant transfected cells. Although both transfected cell lines contain FGF‐1 cell surface receptors as judged by crosslinking studies, the wild‐type transfects are refractory to exogenous FGF‐1, whereas the mutant transfectants respond normally. Together these results support an intracellular role of wild‐type FGF‐1 in mediating certain of its functions. In addition, they demonstrate that certain functions of the growth factor can be dissociated at the structural level. Additional mutagenesis studies have resulted in the identification of mutants with heparin‐binding or mitogenic deficiencies that do not correlate as well as those of the 132 mutant. It appears that the inactivity of the lysine 132 mutant is related, in part, to cysteine 131. © 1994 Wiley‐Liss, Inc.
Heparan sulfate fibroblast growth factor receptor complex: Structure‐function relationships
Authors: McKeehan Wallace L. , Kan Mikio
Published: February 2005

Splice variations in genes coding for the transmembrane FGF receptor (FGFR) result in isoforms that vary in the ectodomain, intracellular juxtamembrane domain, and the intracellular kinase domain. An analysis of biochemical functions of distinct recombinant isoforms expressed in baculoviral‐infected insect cells allowed generation of models for function of splice variants in both the ecto‐ and intracellular domains. A structural model for the ectodomain of the FGFR is proposed as follows. Alternately‐spliced immunoglobulin‐like disulfide Loop I, which is not required for ligand‐binding, is sufficiently interactive with the base FGF binding site formed by Loops II and III to modify ligand affinity and affect interaction of the receptor with heparan sulfate cofactor. The NH2‐terminal domain of Loop II, which is highly conserved across all isoforms, exhibits a 19‐residue heparin‐binding domain which is obligatory for FGF binding. Heparin protects a 30‐kDa ligand‐binding fragment from proteolysis that is composed of Loop II, the inter‐Loop II/III sequence, and the NH2‐terminus of Loop III. This suggests that the high‐affinity FGF receptor complex is an intimate ternary complex of transmembrane tyrosine kinase, heparan sulfate glycosaminoglycan, and FGF, each of which have interactive binding domains for the other and may contribute to specificity of the FGFR complex. Although Ig Loop II, the inter‐Loop II/III sequence, and the NH2‐terminus of Loop III with heparan sulfate form the base FGF binding site, mutually exclusive alternate splicing of two exons coding for the COOH‐terminal half of Loop III determines which specific members of the FGF ligand family bind with high affinity to the base site.

A kinase‐ and tyrosine phosphorylation site‐defective splice variant, FGFR type 2, acts as a dominant‐negative suppressor of phosphorylation of specifically tyr‐653 in the catalytic domain of the kinase, with less effect on phosphorylation of tyr‐766 in the COOH‐terminal tail. We propose that phosphorylation of tyr‐766, which is required for interaction of phospholipase Cγ1 (PLCγ1) with the receptor, may occur by a cis‐intramolecular mechanism within FGFR monomers, while phosphorylation of tyr‐653, which is required for phosphorylation of PLCγ1, may occur by a trans‐intermolecular mechanism between monomers within kinase homodimers. From the combined results, we propose a model whereby increasing concentrations of FGF may control FGF‐mediated signal transduction by heterodimerization of different FGFR monomers. Different monomers arise by regulated combinatorial alternate splicing that alters both the extracellular and intracellular domains. © 1994 Wiley‐Liss, Inc.
Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm
Authors: Matsumoto Kazuya , Anzai Masayuki , Nakagata Naomi , Takahashi Akio , Takahashi Yumi , Miyata Kenji
Published: February 2005

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken β‐actin promoter‐driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1‐ to 2‐cell stage. RNA transcripts of the luciferase gene were first detected in the 1‐cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon‐count imaging, functional luciferase was identified at the 2‐cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1‐cell embryos, although paternally derived protein is not synthesized until the 2‐cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1‐cell stage. ©1994 Wiley‐Liss, Inc.
Transfer of nuclei from 8‐cell stage mouse embryos following use of nocodazole to control the cell cycle
Authors: J. Otaegui P. , T. O'Neill G. , Campbell K. H. S. , Wilmut I.
Published: February 2005

Mouse 2‐, 4‐, 8‐, and 16‐cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embyros after release from the drug was determined. The minimum effective concentration (95% of arrested nuclei) for 4‐, 8‐, and 16‐cell embryos was 5μM nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P < 0.001) and the length of exposure to nocodazole (P < 0.001). Exposure to any concentration of nocodazole within the range 2.5–10 μM for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5μM nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embyros developing to the blastocyst stage and to term (P < 0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4‐cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60–90 min and started DNA synthesis at 120–150 min after release from nocodazole. Nuclei from blastomeres thus synchronized were used to conduct studies on the effect of the cell cycle on nuclear transfer. A signficant effect was found. When nuclei from 8‐cell embryos in G1 or S‐phase were used as nuclei donors, development to blastocyst was respectively 27% and none. ©Wiley‐Liss, Inc.
Formation of the vitelline envelope precedes the active uptake of vitellogenin during oocyte development in the rainbow trout,Oncorhynchus mykiss
Authors: Johan Hyllner Sven , Silvers Christer , Haux Carl
Published: February 2005

In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. lmmunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 μm. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol‐17β increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 μm) present just inside the oolemma, when the oocytes reached a diameter of 600 μm. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol 17β levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 μm had an immunoreactive vitelline envelope with a thickness of about 3 μm. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol‐17β observed in females are of physiological significance. © 1994 Wiley‐Liss, Inc.
Basal components of the transcription apparatus (RNA polymerase II, TATA ‐binding protein) contain activation domains: Is the repetitive c‐terminal domain (CTD) of RNA polymerase II a “Portable Enhance Domain”?
Authors: Seipel Katja , Georgiev Oleg , Gerber Hans‐Peter , Schhaffner Walter
Published: February 2005

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N‐terminal parts of mammalian TATA‐binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C‐terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA‐binding domain to the N‐terminal domain of human TBP or subdomains of it, and to the C‐terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box (“promoter” position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote (“enhancer”) position.

Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with “proximal” activation domains of promoter‐bound transcription factors. They also imply that the C‐terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer‐bound transcription factors. We suggest that the CTD of RNA polymerase II contains a “portable” remote activation domain that may also facilitate chromatin opening within the transcription unit. © 1994 Wiley‐Liss, Inc.
Transforming growth factor‐β1 in reproduction
Authors: M. Shull Marcia , Doetschman Thomas
Published: February 2005

Expression patterns of TGF‐βs during embryogenesis and in adult reproductive organs, as well as the activities of these molecules in in vitro assays of biological processes relating to reproduction and development, have suggested that TGF‐βs may play a role in both reproductive function and embryonic development. To investigate the function of TGF‐β1 in vivo, the murine TGF‐β1 gene was disrupted by gene targeting, and animals that lacked TGF‐β1 activity were generated. Homozygous mutant animals were obtained which exhibited a multifocal inflammatory disease. However, the observed numbers of homozygous mutant offspring were less than expected, suggesting the occurrence of some type of prenatal lethality. This paper reviews the proposed role of the TGF‐βs in reproductive and developmental processes and discusses observations obtained from the TGF‐β1 gene‐targeting experiments as they relate to these processes. © 1994 Wiley‐Liss, Inc.
Analysis of thymidine kinase gene expression in preimplantation mouse embryos
Authors: Lee Dae Kee , Sun Woong , Cho Hyeseong , Kim Kyungjin , Rhee Kunsoo , Lee Chung Choo
Published: February 2005

Thymidine kinase (TK) activity was examined during the development of preimplantation mouse embryos. TK activity was increased approximately 20‐fold from day 2 embryos (2‐cell) to day 5 embryos (late blastocyst). TK activity did not change along with the progression into S‐phase of the first and the second cell cycles but increased sharply at S‐phase of the third cell cycle. Analysis of TK mRNA with a reverse transcription‐polymerase chain reaction (RT‐PCR) method showed that the level of TK mRNA was low in ovulated eggs and 1‐cell embryos and was hardly detectable in day 2 embryo (2‐cell), but sharply increased in day 3 embryos (mixture of 5‐ to 8‐cell and morula). The functional role of 5′‐flanking sequence of TK gene was also investigated in preimplantation embryos after microinjection with the DNA construct of 5′‐flanking sequence of TK (2.4 kb) linked to bacterial lacZ gene (TK2.5lacZ) into the pronucleus of 1‐cell and subsequently by histochemical staining with X‐gal. β‐Galactosidase activity was first detected in day 3 embryos (8‐cell), and 30% of embryos were stained with X‐gal in day 4 and day 5 embryos, respectively. These results show that an increase in TK activity occurred after 2‐cell stage, and this increase was primarily due to the embryonic activation of TK gene expression. Also, it appears that the 5′‐flanking sequence of TK may directly regulate the TK gene expression at the transcriptional level during preimplantation murine development. © 1994 Wiley‐Liss, Inc.
Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm
Authors: Saragüeta Patricia , Lanuza Guillermo , V. Miranda Patricia , G. Tezón Jorge , Lino Baraño J.
Published: February 2005

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR‐inducing activity. AR activity was associated with a protein fraction of Mr > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent Mr 50,000 and 29,000. The N‐terminal sequences of these bands showed a 100% homology with the heavy and light chains of human lgG. A polyclonal antibody raised against the purified protein and anti‐human lgG were both able to suppress the acrosome reactioninducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human lgG were able to mimic the AR‐inducing activity of hFF. We concluded that the AR‐inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies. © 1994 Wiley‐Liss, Inc.
Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo‐3. inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: Tyrphostin A48, pertussis toxin, and 3‐quinuclidinyl benzilate
Authors: L. Bailey Janice , T. Storey Bayard
Published: February 2005

The fluorescent calcium indicator, fluo‐3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25°C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F‐127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]j. Initial [Ca2+]j was 231 ± 58 nM (±SE, n = 43). Addition of heat‐solubilized mouse zonae pellucidae to capacitated sperm increased [Ca2+]j by 106 ± 19 nM (±SE, n = 18), the higher steady‐state concentration being reached after 30 min. Subsequent addition of the non‐fluorescent calcium ionophore Br‐A23187 resulted in a further increase of 114 ± 18 nM (± SE, n = 18), the higher steady‐state concentration being reached after 6 min. The increase in [Ca2+]j induced by solubilized zonae pellucidae was largely blocked by 3‐quinuclidinyl benzilate (QNB) an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121–130). This [Ca2+]j increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida‐induced increase in [Ca2+]j all three inhibitors also blocked the zona pellucidainduced acrosome reaction. These results indicate that [Ca2+]j increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca2+]j suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding. © 1994 Wiley‐Liss, Inc.
Developmental expression of poly(A) binding protein mRNAs during spermatogenesis in the mouse
Authors: C. Kleene K. , Wang M‐Y. , Cutler M. , Hall C. , Shih D.
Published: February 2005

The poly(A) binding protein (PABP), a conserved protein that binds to the 3′ poly(A) tail on mRNAs in eukaryotic cells, has been implicated in the regulation of mRNA stability and translation. Two PABP cDNAs with different sequences were isolated from mouse testis cDNA libraries. The predicted amino acid sequence of one, PABP1, is nearly identical (98.9%) to human liver PABP, while 80% of the amino acids of the second, PABPt, are identical to mouse and human PABPs. Northern blots reveal that there is one major PABP mRNA species in liver, muscle, kidney, and brain, two in spleen, and at least four in testis. The levels of PABP mRNA in testis are 5–10‐fold higher than in these somatic tissues, but surprisingly the vast majority of all PABP mRNA size variants sediment more slowly than single ribosome, indicating strong translational repression. Reverse transcriptase‐polymerase chain reaction assays demonstrate that PABPt mRNAs are abundant only in testis. Northern blots of RNAs purified from highly enriched spermatogenic cells show that the high levels, multiple sizes of PABP mRNAs, and the PABPt mRNA are present in meiotic and early haploid spermatogenic cells, and are sharply reduced in late haploid cells. Comparison of the binding of PABP1 and PABPt to poly(A) Sepharose in vitro revealed subtle differences, even though PABPt contains substitutions for highly conserved aromatic amino acids that are thought to be necessary for binding to poly(A). The existence of two PABP isoforms in mouse spermatogenic cells could influence cytoplasmic gene expression during spermatogenesis. © 1994 Wiley‐Liss, Inc.
Timetable of in vivo embryonic development in the grey short‐tailed opossum (Monodelphis domestica)
Authors: E. Mate Karen , Robinson Edward S. , A. Pedersen Roger , L. Vandeberg John
Published: February 2005

The timing of development was examined in 496 embryos from female Monodelphis domestica, collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three‐celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two‐cell stage. The four‐cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96–100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.© 1994 Wiley‐Liss, Inc.
Cleavage and 3H‐uridine incorporation in bovine embryos of high in vitro developmental potential
Authors: Plante Louise , L. Shepherd Dena , Allan King W. , Plante Claire
Published: February 2005

The timing of genome activation in bovine embryos is still not well defined. The objective of this study was therefore to investigate transcription in bovine embryos with a high potential to develop in culture after in vitro fertilization, by examining, autoradiographically, their incorporation of 3H‐uridine. Initial experiments determined that developmental potential in vitro could be related to the time of first division of the zygote. Embryos that completed their first cleavage within 30 hours of exposure to sperm were more likely to develop into blastocysts (65.7%) and to hatch (50.9%). Using such embryos, it was found that 10 of 12 8‐cell and all 11 4‐cell stage embryos were labeled after a 2–4‐hr exposure to 3H‐Uridine. Among 2‐cell stage embryos, 0 of 23, 3 of 17, 8 of 15, and 3 of 4 were labeled after exposure to 3H‐uridine of 2, 4, 7, and 10 hr, respectively. Treatment with α‐amanatin (10–100 m̈g/ml) blocked 3H‐uridine incorporation but did not inhibit cleavage during the first 4 cell cycles. It was concluded that transcription occurs as early as the 2‐cell stage in bovine embryos in vitro but is not critical to the first four cell cycles. © 1994 Wiley‐Liss, Inc.
Implication that potassium flux and increase in intracellular calcium are necessary for the initiation of sperm motility in salmonid fishes
Authors: Tanimoto Satomi , Nakazawa Tohru , Kudo Yoshihisa , Morisawa Masaaki
Published: February 2005

Flux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K+‐ free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subseqent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility. © 1994 Wiley‐Liss, Inc.
Differential localization of two isoforms of the regulatory subunit RIIα of cAMP‐dependent protein kinase in human sperm: Biochemical and cytochemical study
Authors: Pariset Claude , Weinman Serge
Published: February 2005

In the present study, immunogold labeling of ultrathin sections of ejaculated sperm was used to obtain insight into the ultrastructural localization and presumable function of type II cAMP‐dependent protein kinase in sperm motion. In the flagellum, a human‐specific isoform of the RIIα subunit was located on the axonemal microtubule wall, whereas a different isoform of broader specificity was present in the cytoplasm at the periphery of the coarse fibers and fibrous sheath. This isoform was also found in the mitochondria. The human‐specific RIIα subunit is likely linked to microtubules by a unique binding protein of Mr 72kD. These findings are in agreement with the concept of a concerted mechanism involving phosphorylation of both the axonemal microtubules and the fibrous structures for the regulation of mammalian sperm motion. © 1994 Wiley‐Liss, Inc.