bluuebunny/crossref_metadata_2025_split huggingface dataset:
Translational activity of mouse protamine 1 messenger ribonucleoprotein particles in the reticulocyte and wheat germ cell‐free translation systems
Authors: Kleene Kenneth C. , Smith Jean 
Published: February 2005

AbstractProtamine 1 mRNAs are inactivated by a block to the initiation of translation in early spermatids and are translationally active in late spermatids in mice. To determine whether translation of protamine 1 mRNAs is inhibited by a protein repressor, the translational activity of ribonucleoprotein particles and deproteinized RNAs were compared in the reticulocyte and wheat germ cell‐free translation lysates. To isolate RNPs, cytoplasmic extracts of total testes were fractionated by large‐pore gel filtration chromatography. Ribonucleoprotein particles in the excluded fractions stimulated synthesis of radiolabeled translation products for protamine 1 about twofold less effectively than deproteinized RNAs in the reticulocyte lysate, but were inactive in the wheat germ lysate. The ability of translationally repressed protamine 1 ribonucleoprotein particles to form initiation complexes with 80S ribosomes in the reticulocyte lysate was also measured. Protamine 1 ribonucleoprotein particles isolated by gel filtration and in unfractionated cytoplasmic extracts of early spermatids were nearly as active in forming initiation complexes as deproteinized mRNAs. The isolation of ribonucleoprotein particles in buffers of varying ionic strength, protease inhibitors, and several other variables had no major effect on the ability of protamine 1 ribonucleoprotein particles to form initiation complexes in the reticulocyte lysate. These results can be explained by artifacts in the isolation or assay of ribonucleoprotein particles or by postulating that protamine 1 mRNAs are inactivated by a mechanism that does not involve protein repressors, such as sequestration. © 1994 Wiley‐Liss, Inc.
Fine structure of equine oocytes matured in vitro for 15 hours
Authors: Willis P. , Caudle A. B. , Fayrer‐Hosken R. A. 
Published: February 2005

AbstractTransmission electron microscopy (TEM) was used to evaluate the fine structure of equine oocytes cultured in vitro. Oocytes obtained by follicular aspiration were cultured for either zero or 15 hr. After treatment oocytes were processed either by light microsocopy (nuclear evaluation) or TEM (cytoplasmic evaluation). Those oocytes cultured for 15 hr were incubated in modified TCM 199 with 15% (v/v) mare serum (day of ovulation) at 39 ± 0.2°C. Evaluation using TEM revealed that cortical granules were present in all oocytes. However, zero‐time oocytes contained few cortical granules, and these were scattered throughout the cytoplasm, whereas 15 hr oocytes contained numerous cortical granules primarily found in very close proximity to the oolemma. Further ultrastructural analysis of both groups revealed organelle structure similar to that previously described for in vivo matured equine oocytes. Evaluation of nucelar maturity (lacmoid stain) showed that 15 hr of culture resulted in significant numbers of oocytes at metaphase II (8/17; 47%). These data demonstrate that oocytes cultured for 15 hr in modified TCM 199 with 15% mare serum (day of ovulation) are mature with respect to nuclear configuration and cortical granule migration and, therefore, would be appropriate candidates for in vitro fertilization. © 1994 Wiley‐Liss, Inc.
Chromatin condensation in hamster sperm: A flow cytometric investigation
Authors: Yossefi Sara , Oschry Yitzchak , Lewin Lawrence M. 
Published: February 2005

AbstractIn this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine. © 1994 Wiley‐Liss, Inc.
Expression patterns of novel genes during mouse preimplantation embryogenesis
Authors: Temeles Gretchen L. , Ram Prahlad T. , Rothstein Jay L. , Schultz Richard M. 
Published: February 2005

AbstractLittle is known about the repertoire of genes expressed following zygotic gene activation, which occurs during the two‐cell stage in the mouse. As an initial attempt to isolate novel genes, we used previously prepared two‐cell and two‐cell subtraction cDNA libraries (Rothstein et al., Genes Dev 6:1190–1201, 1992) to isolate a panel of seven cDNA clones. Three cDNA had no match in the current DNA sequence data banks and three others revealed sequence homology to portions of sequences in the data banks. One cDNA was 90% homologous to the ras‐related gene Krev/rap 1A. The temporal patterns of expression of these genes during oocyte maturation and preimplantation development were analyzed by a reverse transcription‐polymerase chain reaction (RT‐PCR) assay developed to measure relative levels of mRNAs. Three distinct temporal patterns of expression, designated Classes 1–3, were found. The two Class 1 genes displayed an actin‐like pattern, with a gradual decline in expression during oocyte maturation and through the two‐cell stage, followed by increases at the eight‐cell and/or blastocyst stages. The four genes in Class 2 were expressed at relatively high levels during oocyte maturation and through the one‐cell stage and then declined abruptly between the one‐ and two‐cell stages; an increase then occurred at the eight‐cell and/or blastocyst stages. The expression of the gene in Class 3 declined during oocyte maturation, but then showed a transient increase at the one‐cell stage, with only a very slight increase in synthesis at either the eight‐cell or blastocyst stage. © 1994 Wiley‐Liss, Inc.
Rat epididymis‐specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis
Authors: Moore Alison , Ensrud Kathy M. , White Thomas W. , Frethem Chris D. , Hamilton David W. 
Published: February 2005

AbstractMonoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent MrS of 38–26 kD, whereas the memrane‐associated form of the molecule has an Mr of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley‐Liss, Inc.
Distribution of prosome proteins and their relationship with the cytoskeleton in oogenesis of Xenopus laevis
Authors: Ryabova L. V. , Virtanen I. , Olink‐Coux M. , Scherrer K. , Vassetzky S. G. 
Published: February 2005

AbstractThe presence of prosome proteins (p25K and p27K) was shown and their distribution was studied in oogenesis of Xenopus laevis using immunoblotting and immunofluorescence. These proteins form numerous granular clusters of variable size all over the cell. At previteilogenic stages, the prosome antibodies homogeneously stain the oocyte nucleus and the evenly distributed relatively large clusters in the cytoplasm. As the oocyte grows, the pattern of distribution of the prosome proteins undergoes changes: animal‐vegetal and cortical gradients appear in the cytoplasm. In the course of oocyte maturation the size of clusters diminishes. Artificial activation of the egg leads to a dorso‐ventral gradient in distribution of the prosome proteins. In this way, specific localization of prosome proteins is first visualized during formation of the dorso‐ventral polarity. Co‐localization of prosome proteins and actin and myosin was found in the oocyte by double staining. Small clusters of prosomes dispersed in the cytoplasm acquire capability of movement (after artificial activation) due, in all likelihood, to persisting connection with the acto‐myosin complex of the egg. © 1994 Wiley‐Liss, Inc.
Molecular cloning and developmental expression of an mRNA encoding the 27 kDa outer dense fiber protein of rat spermatozoa
Authors: Morales C. R. , Oko R. , Clermont Y. 
Published: February 2005

AbstractWe have isolated a cDNA (ODF27), encoding the major 27 kDa protein of rat sperm outer dense fibers (ODF), by screening a testicular lambda‐gt11 phage cDNA library with an affinity‐purified anti‐27 kDa ODF polyclonal antibody. A cyanogen bromide derived internal amino acid (a.a.) sequence of the 27 kDa ODF protein was identical to an internal region of the deduced a.a. sequence of this cDNA. The cDNA encodes a protein with a high proportion of a repetitive motif, Cys‐Gly‐Pro, at the carboxy‐terminal end, reminiscent of the testis‐specific Mst(3)CGP proteins of Drosophila melanogaster (Schäfer et al., 1993. Mol Cell Biol 13:1708–1718). Nick translation probes of the ODF27 cDNA recognized two complementary mRNAs of 1.2 and 1.5 kb in the rat testis. Developmental Northern blot analysis revealed that these mRNAs are first transcribed in round spermatids. In situ hybridization confirmed the haploid expression of these transcripts and demonstrated that they are found in the cytoplasm of spermatids throughout most of the duration of spermiogenesis. They reach a peak in steps 8–10 of spermiogenesis at the time transcription ceases, remain at high levels from steps 11 to 15, and diminish in steps 16–18 at the time ODF protein synthesis and assembly are shown to be maximum. The translation of these transcripts, therefore, appears to be post‐transcriptionally controlled. A literature and NCBI database search revealed that the nucleotide sequence of the 027 cDNA is homologous to the rat gene RT7 (Van Der Hoorn et al., 1990. Dev Biol 142:147–154) and to the rat testis‐specific cDNA rts 5/1 (Burfeind and Hoyer‐Fender, 1991. Dev Biol 148:195–204), which encodes a 27 kDa polypeptide. © 1994 Wiley‐Liss, Inc.
Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling
Authors: Mate Karen E. , Ksower Nechama S. , White Ian G. , Rodger John C. 
Published: February 2005

AbstractThe acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane‐impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane‐permeable agent mBBr lightly labelled the perimeter of the acrosome of non‐DTT‐treated possum and wallaby spermatozoa, indicating the presence of peri‐acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane‐impermeable agent qBBr did not label any part of the acrosome in non‐DTT or DTT‐treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT‐treated spermatozoa. The nucleus was not labelled in non‐DTT or DTT‐treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley‐Liss, Inc.
cDNA cloning and expression of Xenopus sperm‐specific basic nuclear protein 5 (SP5) gene
Authors: Ariyoshi N. , Hiyoshi H. , Katagiri H. , Abé S.‐I. 
Published: February 2005

AbstractAs part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm‐specific nuclear proteins (SPs1–6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)+ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N‐terminal regions of all the SPs(1–6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one‐fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0. © 1994 Wiley‐Liss, Inc.
Developmentally regulated testicular galactolipid sulfotransferase inhibitor is a phosphoinositol glycerolipid and insulin‐mimetic
Authors: Lingwood Clifford A. , Sakac Darinka , Saltiel Alan 
Published: February 2005

AbstractThe synthesis of sulfogalactosyl‐glycerolipid (SGG) is a differentiation marker in spermatogenesis restricted to the zygotene and early pachytene spermatocytes. The galactolipid sulfotransferase responsible for the synthesis of SGG is regulated by a phosphorylation mechanism. The activity of this enzyme is reduced in cells later in spermatogenesis by a low molecular weight inhibitor, which can be extracted in organic solvents and purified by reverse phase high pressure liquid chromatography (HPLC). This purified inhibitor is a potent postreceptor insulin‐mimetic, which stimulates adipocyte lipogenesis more effectively than does insulin. Phosphoinositol (PI) glycolipids have been proposed as second messengers of the insulin phosphorylation cascade. These species contain a nonacetylated glucosamine, which renders them liable to cleavage by deamidation. The activity of the sulfotransferase inhibitor was lost following nitrous acid deamidation and was labile to PI specific phospholipase C digestion. Insulin and insulin‐like growth factor I were found to inhibit germ cell synthesis of SGG in vitro to some degree but had no direct effect on the testicular galacto‐lipid sulfotransferase assay. These results indicate that the sulfotransferase inhibitor is a glycosyl phosphoinositide similar to the lipid species, which mediate insulin signal transduction and suggest that germ cell SGG biosynthesis may be regulated by a receptor‐mediated phosphorylation pathway. © 1994 Wiley‐Liss, Inc.
Extracellular matrix abnormalities in testis and epididymis of XXSxr(“sex‐reversed”) mice
Authors: Griffin Jennifer K. , Blecher Stan R. 
Published: February 2005

AbstractSex‐reversed (Sxr) is a duplication of the sex‐determining region of the Y chromosome, which gets transposed to a paternal X chromosome. Chromosomally female (XX) zygotes that receive this XSxrchromosome develop as apparent males. Previous work on XXSxr mice (called pseudomales) showed extracellular matrix (ECM) ultrastructural abnormalities in the epididymis and testis. This study examined the biochemical nature of these abnormalities. More hydroxyproline (an indicator of collagen) was noted in the pseudomale testis and epididymis compared to normal male tissues. Western blot analysis showed increased collagen IV in the pseudomale testis and epididymis. In both the hydroxyproline and collagen IV studies, the epididymis was found to contain higher levels of these substances than the testis for both genotypes. There also appeared to be increased messenger RNA for tissue inhibitor of metalloproteinases (Timp), a regulator of collagen, in the pseudomale testis. Data from these studies seem to indicate that the XXSxr genotype influences ECM deposition and/or turnover and exerts a direct genetic influence on the development of the testis and epididymis. According to the existing paradigm of mammalian sexual development, the epididymis is expected to be normal in the presence of adequate androgenization and independent of chromosomal and genetic sex. The results presented here differ from what would be predicted by this paradigm. © 1994 Wiley‐Liss, Inc.
Expression of activin receptor II and IIB mRNA isoforms in mouse reproductive organs and oocytes
Authors: Wu Tsung‐Chieh J. , Jih Ming H. , Wang Lai , Wan Yu‐Jui Y. 
Published: February 2005

AbstractActivins, the dimeric polypeptides of inhibin β‐subunits, exhibit paracrine effects on cell proliferation, differentiation, and various other cell functions. The complex biological response to activin appears to involve multiple receptors. In the present study, we examined the isoform mRNA expression of both activin receptor type II (ActR‐II) and type IIB (ActR‐IIB) genes in mouse reproductive organs, cumulus–oocyte complexes (COCs), and ovulated oocytes. Northern blot analyses of female and male reproductive organs with single‐stranded ActR‐II cDNA probes revealed that mouse ovaries expressed high levels of the 6.0 kilobase (kb) mRNA, whereas the 3.0 kb transcript was the major mRNA species found in the testis. Reverse transcriptase–polymerase chain reaction (RTPCR) showed that both COCs and oocytes contained ActR‐II mRNA. To examine the expression of ActR‐IIB gene, primer selection was made outside the two alternative splicing sites in order to amplify the cDNAs of all four distinct receptor isoforms. The results of RT‐PCR demonstrated that isoforms IIB2 and IIB4 were the major mRNA species expressed in both female and male gonads and extragonadal reproductive tissues. The ovary expressed all four mRNA isoforms, whereas the testes expressed only three isoforms. Furthermore, COCs and oocytes contained only the ActR‐IIB2 isoform. The differential expression of both activin receptor mRNA isoforms in the reproductive organs suggests that distinct alternative splicing mechanisms are involved in activin receptor gene expression in male and female gonads, and that each of the activin receptors may have its own biological function in reproduction. The expression of activin receptor genes in COCs and oocytes indicates that activin, produced by the granulosa cells of maturing follicles, may act locally to regulate follicular development and oocyte maturation. © 1994 Wiley‐Liss, Inc.
Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism
Authors: Armstrong V. L. , Clulow J. , Murdoch R. N. , Jones R. C. 
Published: February 2005

AbstractThe role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25–30 nl of CEF was stimulated by calcium ions (Ca2+), N,2′ ‐O‐dibutyryl‐guanosine 3′:5′ ‐cyclic monophosphate (dibutryl cGMP), cyclic adenosine 3′:5′‐monophosphate (cAMP), N6,2′‐O‐dibutyryladenosine 3′:5′ ‐cyclic monophosphate (dibutyryl cAMP), 8‐bromoadenosine 3′:5′ ‐cyclic monophosphate (8‐bromo cAMP), caffeine, theophylline and bicarbonate ions (HCO3−). Other agents such as magnesium ions (Mg2+), veratridine, phospholipase C (PLC), ionophore A23187, 1,2‐dioctenoyl‐sn‐glycerol (DAG), phorbol 12‐myristate 13‐acetate, phospholipase A2 (PLA2), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D‐[U‐14C]glucose and [1‐14C]acetate as exogenous energy sources for oxidative metabolism. No detectable 14C‐lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca2+ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA2 pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose‐carbon through the glycolytic pathway. © 1994 Wiley‐Liss, Inc.
Rapid, nonradioactive, and quantitative method to analyze zona pellucida modifications in single mouse eggs
Authors: Moos Jiri , Kalab Petr , Kopf Gregory S. , Schultz Richard M. 
Published: February 2005

AbstractA rapid, nonradioactive method to monitor the ZP2 to ZP2f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2f conversion following A23187‐induced egg activation, when compared to the commonly used radioiodination procedures. © 1994 Wiley‐Liss, Inc.