bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractWhile 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

AbstractThe 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

AbstractInsulin‐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

AbstractThe 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

AbstractBovine 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

AbstractChromatin 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.
Insulin regulates protein metabolism in mouse blastocysts
Authors: Dunglison Greta F. , Kaye Peter L. 
Published: February 2005

AbstractMouse blastocysts, in vitro, endocytosed 100 μg/ml 125I‐labelled bovine serum albumin (BSA) at a rate equivalent to 192 ± 27 μl/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of 125I‐labelled BSA began to plateau as the endocytosed 125I‐labelled BSA was catabolized and 125I was released from the cells. Insulin caused an ≈︁72% (P < 0.05) increase in the amount of uncatabolized 125I‐labelled BSA remaining in insulin‐treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed 125I‐labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin‐treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin‐treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short‐term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein‐reserves in the embryo. Dose‐response studies indicated an EC50 of 0.5 pM for insulin's stimulation of 125I‐labelled BSA accumulation, consistent with action via its own receptor. Insulin‐like growth factor‐1 (IGF‐1) also stimulated protein accumulation at concentrations similar to those observed with insulin, suggesting that IGF‐1 may act via its own receptor rather than the insulin receptor to exert its effects on endocytosis. © 1993 Wiley‐Liss, Inc.
Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte‐cumulus cell complexes in vitro
Authors: Singh Balwant , Barbe Gerald J. , Armstrong David T. 
Published: February 2005

AbstractThe present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor‐b̃1 (TGF‐b̃1), follicle‐stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A4), and estradiol‐17b̃ (E2) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte‐cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E2 (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A4 (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF‐b̃1 had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley‐Liss, Inc.
Embryonic stem cells derived from morulae, inner cell mass, and blastocysts of mink: Comparisons of their pluripotencies
Authors: Sukoyan Marina A. , Vatolin Sergei Y. , Golubitsa Alevtina N. , Zhelezova Antonina I. , Semenova Lora A. , Serov Oleg L. 
Published: February 2005

AbstractA characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two‐thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst‐derived lines had one of the Xs in an inactive state. The activity of glucose‐6‐phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst‐derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae‐ and ICM‐derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM‐ or morula‐derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley‐Liss, Inc.
Evidence for the production of a growth‐inhibitory factor by human granulosa‐luteal cells
Authors: Dain Liliana B. , Bley Miguel A. , Barañao José L. , Tesone Marta 
Published: February 2005

AbstractThe factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle‐stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8‐fold within a 2‐day period. In serum‐free medium, human luteinized granulosa cells were able to incorporate 3H‐thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, 3H‐thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH‐ and estradiol‐treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze‐thawing, was stable for several weeks at – 20°C, became unstable at 4°C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight >30,000 dalton. We suggest that an inhibitory growth factor produced by human luteinized granulosa cells could be involved in the differentiation of growing follicles to corpus luteum. © 1993 Wiley‐Liss, Inc.
Nuclear transfer and electrofusion in bovine in vitro‐matured/in vitro‐fertilized embryos: Effect of media and electrical fusion parameters
Authors: Van Stekelenburg‐Hamers Annelies E. P. , Van Inzen Wouter G. , Van Achterberg Tanja A. E. , Kruip Theo A. M. , De Laat Siegfried W. , Weima Sjerp M. 
Published: February 2005

AbstractIn this study, micromanipulation and electrofusion conditions for the cloning of in vitro‐produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro‐matured (IVM) and in vitro‐fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 μsec. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle. © 1993 Wiley‐Liss, Inc.