bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and preliminary characterization of guinea pig testicular proteinase inhibitors
Authors: Falase E. A. O. , Storey B. T. , Teuscher C. 
Published: February 2005

AbstractThree guinea pig testicular, low‐molecular‐weight, acid‐stable inhibitors specific for typsin‐like proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G‐100 and G‐50, ion‐exchange chromatography on SP‐Sephadex, followed by QAE‐Sephadex. Final purification was by rechromatography on Sephadex G‐50 superfine gel. The three proteinase inhibitors were labeled A, B, and Cnb, the latter to denote nonbinding of Cnb to the QAE‐Sephadex. Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin‐catalyzed hydrolysis of the N‐benzyloxycarbonyl‐L‐arginyl amide of 7‐amino‐4‐trifluoro‐methylcoumarin (CBZ‐Arg‐AFC) at pH 8.0. The values were calculated to be, for A, 1.5 × 10−8 M; for B, 1.5 × 10−8 M; and, for Cnb, 2.2 × 10−7 M. The Ki values calculated from inhibition of trypsin‐catalyzed hydrolysis of the active site titrant 4‐methylumbelliferyl‐p‐guanidinobenzoate (MUGB) using Easson‐Stedman plots were, for A, 7.7 × 10−9 M; for B, 6.7 × 10−9 M; and, for Cnb, 1.4 × 10−7 M. The MrS as determined by active site titration with MUGB were A, 11.2 kDa; B, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gave Mr values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for A and 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for B indicates that it may function as a dimer or trimer in the active state.
3H‐uridine incorporation in early porcine embryos
Authors: Freitag M. , Döpke H. H. , Niemann H. , Elsaesser F. 
Published: February 2005

AbstractThe present study investigated the ontogeny of 3H‐uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two‐cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 μM 3H‐uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two‐cell stage. From the four‐cell to the morula stage, 3H‐uridine incorporation per embryo increased about ninefold (P < 0.001) in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H‐uridine incorporation was not different from the four‐cell stage up to the zona pellucida‐intact blastocyst and amounted to 0.29–0.37 fmol 3H‐uridine incorporation/cell/3 hr. In hatched blastocyst, 3H‐uridine incorporation per blastomere was increased (P < 0.01 compared with younger stages) and amounted to 0.86 fmol 3H‐uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida‐intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.
Analysis of proteins synthesized by 9.5 day mouse embryos: Determination of cardiac and noncardiac proteins
Authors: Van Keuren Margaret L. , Iacob Ruxandra A. , Kurnit David M. 
Published: February 2005

AbstractTo catalog polypeptides that were specific to developing hearts, we separated 35S‐methionine‐labeled 9.5 day mouse embyros into cardiac and noncardiac (carcass) components. Two‐dimensional gels were then used to analyze the polypeptides synthesized in these two fractions. As a result, we were able to distinguish polypeptides that were specific to or increased in the heart as well as those polypeptides that were specific to or inceased in the embryo minus the dissected heart. Using this analysis, there were two polypeptides that were cardiac‐specific and 17 that were expressed at increased levels by at least twofold in the heart. The cardiac‐specific polypeptides may be used in further studies to identify early cardiac tissue. Conversely, there were 26 polypeptides unique to noncardiac structures and an additional 15 that were increased in the carcass more than twofold relative to the heart. The noncardiac‐specific polypeptides may be used to define contamination of putative cardiac tissue with noncardiac material. Two of the polypeptides expressed more abundantly in the carcass appeared to correspond to known proteins in the mouse fibroblast database, cyclin and tropomyosin 4. Thus the heart at 9.5 days of murine development can be distinguished readily from the remainder of the embryonic mouse both macroscopically and on two‐dimensional gels.
Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus
Authors: Borsuk Ewa 
Published: February 2005

AbstractEight‐h‐old anucleate fragments of artificially activated mouse oocytes produced within 30 min after activation were fused with anucleate fragments of ovarian oocytes bisected at the GV stage and matured in vitro for 4–5 h. The resulting cybrids were inseminated soon after fusion and fixed 3–4 h or 14 h later. After 3–4 h of culture most of the sperm nuclei underwent decondensation. Early male pronuclei were observed sporadically. After overnight culture small or fully formed male pronuclei were present in monospermic and dispermic cybrids. The abortive nuclei, designated as stage lla (earlier described by Borsuk and Tarkowski, 1989) were also observed and their occurrence was strongly correlated with the rate of polyspermy. In unfused (control) anucleate fragments of maturing and activated oocytes inseminated at the same time as the cybrids, male pronuclei were never observed. These results show that the cytoplasms of activated eggs and of maturing oocytes contain complementary factors required for the transformation of the sperm nucleus into the male pronucleus. The former has lost the capability to break down the sperm nucleus envelope but contains factors required for pronuclear growth. The latter is able only to initiate the process of transformation, i.e., to break down the nuclear envelope and decondense the denuded sperm chromatin. However, the activity of the factor responsible for nuclear envelope breakdown is limited because in cybrids penetrated by more than two spermatozoa, transformation of sperm nuclei was usually abnormal.
Cytochalasin D treatment induces meiotic resumption in follicular sheep oocytes
Authors: De Smedt V. , Szöllösi D. 
Published: February 2005

AbstractOvine cumulus‐enclosed oocytes collected from antral follicles (3–5 mm in diameter) were cultured in vitro with 2 ± 106 granulosa cell/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle‐stimulating hormone‐luteinizing hormone (FSH‐LH; 10 μg/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6–7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%) remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus‐oocyte complexes (COCs) were incubated in CD (5 μg/ml) for 6 h at the beginning of the culture. Under these conditions, 85% of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte.
Role of the extracellular matrix in tissue‐specific gene expression in the sea urchin embryo
Authors: Benson Steve , Rawson Robert , Killian Christopher , Wilt Fred 
Published: February 2005

AbstractThe role of extracellular matrix (ECM) in the differentiation of tissue types was examined in embryos of Strongylocentrotus purpuratus. We have examined the expression of various tissue‐specific molecular markers after disrupting the ECM by culturing embryos in the presence of β‐aminoproprionitrile fumarate (BAPN), which disrupts collagen deposition, and β‐D‐xyloside, which disrupts proteoglycan metabolism. The markers examined included accumulation of primary mesenchyme‐specific mRNA (SM 50); and aboral ectoderm‐specific mRNA (Spec 1); and a gut‐specific enzyme, alkaline phosphatase. Treatment with BAPN or β‐D‐xyloside results in developmental arrest at the mesenchyme blastula stage. Although spicule formation is inhibited, the accumulation of SM 50 transcripts and the synthesis of most of the prominent spicule matrix proteins is similar to that of control embryos. Spec 1 mRNA, in contrast, while accumulating to a significant extent when collagen and proteoglycan metabolism is disrupted, does accumulate to a level somewhat lower than that seen in control embryos. Additionally, the postgastrula rise in gut‐specific alkaline phosphatase is reversibly inhibited by BAPN and xyloside treatment. These results demonstrate a differential effect of the ECM on expression of tissue‐specific molecular markers.
Unique 3′‐untranslated sequence of insulin‐like growth factor‐I isolated from human placenta
Authors: Daimon M. , Wang C.‐Y. , Johnson T. R. , Ilan Joseph , Ilan Judith 
Published: February 2005

AbstractA cDNA clone of 525 bp corresponding to the 3′‐untranslated region of insulin‐like growth factor‐I was isolated from a human placenta library. The sequence of this clone extended 200 nucleotides downstream from the previously reported 3′‐end of IGF‐IA cDNA, indicating the existence of IGF‐IA transcripts having an even larger 3′‐untranslated region. By using this clone for RNA transfer blot hybridization, it was shown that this longer 3′‐untranslated region is included in the 7.5‐ and 5.0‐kb transcripts, but not in the 1.1‐ and 0.9‐kb transcripts. It is also apparent that transcripts bearing the extended 3′‐untranslated sequence are highly expressed in human placenta.
Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female
Authors: Jeyalectumie C. , Subramoniam T. 
Published: February 2005

AbstractBiochemical studies on the male reproductive tissues and seminal secretions have been made with reference to sperm metabolism and different stages of maturity in the crab Scylla serrata. The results reveal that the seminal plasma and spermatophores are rich in protein, carbohydrate, and lipid. In general, organic components of spermatophores are considerably higher than those of seminal plasma. Enzyme studies show that the succinate dehydrogenase (SDH) activity is very low, whereas fumarate reductase (FR) and lactate dehydrogenase (LDH) exhibit high activity. Electrophoretic studies on LDH show that, in addition to the occurrence of a sperm‐specific fraction, LDHx, the M‐type subunits are predominant in the mature spermatophores. These results from enzyme studies suggest that sperm metabolism is mainly anaerobic, utilizing the carbohydrates as substrates. The results for maturational changes reveal that the male reproductive tissues and their secretions contain lesser quantity of organic components in the immature crabs; as the maturity proceeds, there is not only concentration of organic substances but also an increase in the size of spermatophores. The concentration of biochemical constituents is highest in the proximal vas deferens (PVD), suggesting that the granular seminal plasma as well as the sperm‐agglutinating substance and spermatophoric wall are secreted in this region. The spermatheca of the unmated female crabs are poor in organic constitutents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. During sperm storage in the spermatheca, only the carbohydrates decline steeply. A low activity of SDH, but a moderate level of LDH and a high level of FR activity, is recorded in the spermathecal content of mated crabs, providing further evidence for anaerobic metabolism of sperm during storage in female. A sharp fall in the stored carbohydrates constitutes further evidence in this regard.
Effect of cell‐free synchronous uterine flushings and microsurgery on the development of porcine embryos in vitro
Authors: Martin M. J. , Cantley T. C. , Flowers W. L. , Day B. N. 
Published: February 2005

AbstractExperiment I was designed to determine if cell‐free synchronous uterine flushings contain an embryotoxic substance that is normally screened by the intact zona pellucida. Sixty 4‐cell embryos were allocated to three treatment groups: (1) control embryos (n = 20) were cultured in Modified Kreb's Ringer Bicarbonate medium + 10% bovine calf serum (mKRB‐BCS), (2) UF embryos (n = 20) were cultured in 80% mKRB‐BCS + 20% sterile dialyzed uterine flushings (UF), (3) MicroUF embryos (n = 20) received a microsurgical incision in the zona pellucida and were cultured in 80% mKRB‐BCS + 20% UF. Following 72 h in culture at 37°C under a 90% N2, 5% CO2, and 5% O2 atmosphere, the number of nuclei/embryo and the incidence of protrusion of the trophoblast through the zona pellucida (PTZ) were recorded. Addition of UF had no effect on embryo development. A greater (P > .005) proportion of MicroUF embryos exhibited PTZ as compared to UF and control embryos. Experiment II was devised to further characterize the occurrence of PTZ in Micro porcine embryos. Thirty‐three 4‐ to 10‐cell embryos and 14 morulae were distributed across two treatments: (1) control embryos (n = 16 and 6, respectively) were cultured as described in Experiment I; and (2) micro embryos were treated similarly to MicroUF embryos in Experiment I but were cultured in mKRB‐BCS only. At the onset of PTZ, embryos were immediately fixed and examined. The proportion of embryos exhibiting PTZ was greater (P < .007) for Micro versus control embryos. Both the mean time to PTZ and the number of nuclei present were greater for blastocysts cultured from control as opposed to Micro morulae (P < .0001 and P < .0002, respectively). These results indicate that (a) cell‐free synchronous uterine flushings are not detrimental to embryos with incised zonae and (b) microsurgery increases the incidence of and reduces the time to protrusion of the embryo through the zona pellucida in cultured embryos.
Presence of follicular fluid in the porcine oviduct and its contribution to the acrosome reaction
Authors: Hansen C. , Srikandakumar A. , Downey B. R. 
Published: February 2005

AbstractTwo experiments were conducted to measure the quantity of follicular fluid entering the porcine oviduct following ovulation and to establish its influence on the sperm acrosome reaction in vivo. Prepubertal gilts treated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) were used in both experiments. In experiment 1, each of 64 gilts was assigned at random to one of four treatment groups (n = 16 per group): I (preovulatory), surgery 38 hr post‐hCG; II (ovulatory), (surgery) 42 hr post‐hCG; III (postovulatory), surgery 46 hr post‐hCG; IV (ovulation blocked), surgery 46 hr post‐hCG but also treated with indomethacin (INDO) at 24 hr. At surgery, both follicular and oviductal fluid were collected for determination of volume and progesterone (P4) concentration. In experiment 2, sperm were recovered surgically from the uterine horn, isthmus, and ampulla of gilts at 46 hr post‐hCG either (1) inseminated and non‐INDO‐treated controls (n = 5) or (2) inseminated and INDO‐treated at 24 hr (n = 4). Using P4 as a marker, it was calculated that only 0.51% ± 0.10% of the available follicular fluid was present in the oviduct near the time of ovulation and that this amount had decreased 10–12‐fold 4 hr later. Mean sperm concentration at 46 hr post‐hCG was higher in the uterine horn than in the other two regions (P < 0.05) but the percentage of acrosome‐reacted sperm was greater in the ampulla (P < 0.05). The ampullae of ovulating gilts, i.e., containing follicular fluid, had a greater percentage of acrosome‐reacted sperm than those in which ovulation was blocked by INDO (P < 0.05). The results of these experiments suggest that only a small amount of follicular fluid reaches the oviduct at ovulation and that this fluid has a stimulatory, but probably nonessential, effect on the acrosome reaction.
JunD mRNA expression differs from c‐jun and junB mRNA expression during male germinal cell differentiation
Authors: Alcivar Acacia A. , Hake Laura E. , Kwon Yunhee K. , Hecht Norman B. 
Published: February 2005

AbstractThe members of the jun family of protooncogenes (junB, c‐jun, and junD) share a high degree of sequence homology and function as transcriptional regulators. Here we compare the pattern of junD mRNA expression during spermatogenesis to that of junB and c‐jun (Alcivar et al.: J Biol Chem 265:20160–20165, 1990). junD transcripts are present at high levels in total RNA obtained from both prepuberal and adult intact testes, with the highest levels at stages containing predominantly premeiotic and postmeiotic germ cells. Analyses of cells isolated from testes of 8‐day‐old mice indicate that the level of the 1.8 kb junD mRNA is higher in type B spermatogonia than in type A spermatogonia. In testes of 17‐day‐old mice, the highest junD mRNA levels are detected in preleptotene spermatocytes compared to leptotene/zygotene and prepuberal pachytene spermatocytes. In cells from adult testes, the junD mRNA levels are higher in postmeiotic round spermatids and residual bodies/cytoplasts than in meiotic pachytene spermatocytes. An additional junD transcript of about 1.6 kb is detected in postmeiotic cells. Analyses of polysomal and nonpolysomal RNAs prepared from isolated testicular cells indicate that in early meiotic cell types the junD transcript is more efficiently loaded onto polysomes than in later cell types. In summary, the pattern of expression of junD differs from that of junB and c‐jun during spermatogenesis most notably in that 1) junD mRNA levels do not increase following dissociation of testicular cells and 2) in contrast to the nearly undetectable levels of junB and c‐jun mRNAs in adult postmeiotic testicular cells, high levels of junD mRNAs are seen. The differential expression of these three jun transcripts during development of the male gonad suggests a complex transcriptional regulatory role for the jun family of protooncogenes during spermatogenesis.
Studies on murine embryo‐derived platelet‐activating factor (EPAF)
Authors: Adamson L. M. , Podsiadly B. , Smart Y. C. , Stanger J. D. , Roberts T. K. 
Published: February 2005

AbstractStudies were carried out using the splenectomized mouse bioassay (SMB) to investigate the nature of embryo‐derived platelet‐activating factor (EPAF) and its relationship to synthetic platelet activating factor (PAF). While both C16‐PAF and embryo conditioned media (ECM) induced a significant platelet decline in the SMB at 15 min postinjection, C18‐PAF induced a similar effect at 30 min postinjection. The degree of EPAF activity in ECM was not altered with increasing embryo number from 2 to 40/ml of media. In contrast, PAF (C16/C18 mixture) induced a linear increase in activity with increasing concentration, leading to lethal effects at high concentrations. While EPAF activity was not significantly altered when ECM was diluted 1/1,000, PAF activity was abolished at 1/10 dilution. EPAF in ECM was not inactivated by mouse plasma; however, lipid exracted ECM, like PAF, underwent rapid inactivation in the presence of plasma. Aggregometer studies using horse platelets showed that ECM and lipid‐extracted ECM were unable to induce platelet aggregation, while thin‐layer chromatography (TLC) purified ECM (Rf 0.23) successfully aggregated horse platelets in vitro. Results suggested that EPAF and PAF are not homologous. EPAF might consist of PAF bound to a regulatory carrier molecule and appears to be associated with EPAF‐inhibitor substance(s) in ECM.
Reduction of embryotoxicity by protein in embryo culture media
Authors: Flood Larry P. , Shirley Barbara 
Published: February 2005

AbstractExperiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs–Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free. The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli‐Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two‐cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA‐supplemented media was greater than that in protein‐free media only when the media was greater than that in protein‐free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentrations, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.
Sequence requirements for embryonic transcriptional activation of a gastrula‐specific actin gene in Xenopus laevis
Authors: Brennan Sean M. 
Published: February 2005

AbstractCytoskeletal actin genes undergo developmentally timed transcriptional activation at the gastrula stage of embryonic development in the amphibain Xenopus laevis. To study the regulation of this process, a molecularly marked cloned actin gene has been introduced into living embryos by microinjection, and levels of its transcripts (which are distinct from endogenous actin message) have been measured by RNase protection. In vitro mutagenesis of the marked gene, followed by microinjection and transcriptional analysis of various mutants, has been used to search for gene sequences that participate in accurate transcriptional initiation and developmental control. Deletion mutants containing only 90 nucleotides of upstream sequence undergo correct developmental regulation, while deletion to –33 prevents normal activation of the gene. In the presence of sufficient upstream sequence, an actin–globin fusion gene, containing only 564 nucleotides downstream of the actin gene transcription startsite, is correctly activated. Taken together, these results imply that all sequences necessary for correct temporal regulation reside between –90 and +564 nucleotides, with respect to the transcriptional start site of the actin gene. They further suggest that developmental activation of actin gene transcription may involve either (1) interaction of non‐DNA binding proteins with basal transcription factors, or (2) the concerted action of ubiquitous promoter‐binding factors and factors that interact with downstream regulatory regions.