bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of receptor tyrosine kinases in the rat testis
Authors: Loveland Kate Lakoski , Hedger Mark P. , Risbridger Gail , Herszfeld Daniella , De Kretser David M.
Published: February 2005

Evidence of receptor/ligand interactions that regulate testis cell function was sought in order to broaden the current understanding of the molecular basis of testis cell function. Using reverse transcription and the polymerase chain reaction, we have obtained novel evidence for the expression of three mRNAs encoding receptor tyrosine kinases in the adult rat testis: the platelet‐derived growth factor type A receptor (PDGF‐RA), the basic fibroblast growth factor receptor (flg), and fetal liver kinase 1 (Flk‐1). A 6.8 kb transcript encoding the PDGF‐RA was observed in RNA prepared from testes of rats aged day 5 through adult, with a decline in relative abundance with increasing age after day 17. Analysis of mRNA from isolated cell preparations (day 21 Sertoli cells, adult Leydig cells, round spermatids, and primary spermatocytes) and testes depleted of specific cell types [ethane dimethane sulfonate (EDS)‐treated and cryptorchid] indicated that the Leydig cell was the predominant source of this mRNA in the adult testis. The addition of PDGF‐BB to cultures of highly purified adult rat Leydig cell preparations resulted in a 40% increase in LH‐stimulated testosterone production, confirming a role for this growth factor in regulation of Leydig cell function. These data indicate that the Leydig cell is a principal site of action of PDGF in the testis. © 1993 Wiley‐Liss, Inc.
cis‐Acting DNA elements involved in oocyte‐specific expression of mouse sperm receptor gene mZP3 are located close to the gene's transcription start site
Authors: Lira Sergio A. , Schickler Michael , Wassarman Paul M.
Published: February 2005

We report that cis‐acting DNA elements involved in oocyte‐specific expression of the mouse sperm receptor gene (mZP3) are located close to the gene's transcription start site. Mice bearing a transgene that consists of only 153 nt of mZP3 5′‐flanking region fused to the firefly luciferase gene (153‐ZP3/LUC) expressed the reporter gene in ovary not in a wide variety of tissues; although two of three lines carrying 153‐ZP3/LUC also expressed the transgene in forebrain and hypothalamus. Within the ovaries of transgenic mice, luciferase activity was restricted to growing oocytes. However, levels of luciferase activity in these oocytes were lower than those in oocytes from mice bearing transgenes that contain a larger segment of mZP3 5′‐flanking region (470–6,500 nt) fused to the firefly luciferase gene. Mice bearing a trans‐gene that consists of 470 nt of mZP3 5′‐flanking region and mZP3 intragenic sequences (ZDT) were also analyzed. The presence of mZP3 intragenic sequences did not result in significantly increased levels of firefly luciferase activity in oocytes of mice carrying the ZDT transgene. Overall, these results suggest that as little as 153 nt of mZP3 5′‐flanking region is sufficient to target expression of the firefly luciferase gene to mouse oocytes and that the mZP3 intragenic sequences probably do not contain enhancer elements. Rather, enhancer elements are probably present between — 153 and — 470 nt of the mZP3 5′‐flanking region. © 1993 Wiley‐Liss, Inc.
Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction
Authors: Chang Michael C. , Berkery Diana , Schuel Regina , Laychock Suzanne G. , Zimmerman Arthur M. , Zimmerman Selma , Schuel Herbert
Published: February 2005

Delta‐9‐tetrahydrocannabinol ((−)δ9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP‐55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP‐55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time‐dependent manner. Specific binding of [ H]CP‐55,940 to sperm, defined as total binding displaced by (−)δ9 THC, was saturable: KD 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP‐55,940 to sperm was reduced in a dose‐dependent manner by increasing concentrations of CP‐55,940, (−)δ9 THC, and (+)δ9 THC. The rank order of potency to inhibit binding of [ H]CP‐55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP‐55,940 > (−)δ9THC > (+)δ9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP‐55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley‐Liss, Inc.
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

Protamine 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

Transmission 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

In 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

Little 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

Monoclonal 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

The 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

We 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

The 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

As 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

The 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

Sex‐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

Activins, 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

The 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

A 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.
Mouse preimplantation embryo development in vitro: Effectof sodium concentration in culture media on RNA synthesis and accumulation and gene expression
Authors: Ho Yugong , Doherty Adam S. , Schultz Richard M.
Published: February 2005

Results of previous studies indicate that culture of preipmlantation mouse embryos in SOM medium containing 85 mM NaCl promotes better development in vitro, as well as supporting higher rates of protein synthesis, when compared to culture in SOM containing 125 mM NaCl (Anbari and Schultz, 1993, Mol Reprod Dev 35:24–28; Biggers et al., 1993, Mol Reprod Dev 34:380–390). In the present study we compare the effect of culturing embryos in these 2 media on several aspects of RNA synthesis and gene expression in order to determine whether the reduced development in SOM containing 125 mM NaCI and lower rates of protein synthesis are correlated with decreases in RNA synthesis and stability and changes in gene expression. Although no apparent differences in the metabolism of [3H]uridine to UMP, UDP, and UTP and its incorporation into total RNA are observed when 2‐cell embryos are cultured to the morula stage in either medium, a 20% decrease in the rate of mRNA synthesis is found when embryos are cultured in SOM containing 125 mM NaCl. In addition, pulse‐chase experiments reveal that total mRNA is less stable when the embryos are cultured in SOM containing 125 mM NaCl. Using a reverse transcription‐polymerase chain reaction to assay for changes in the relative amounts of specific mRNAs, the relative amounts of mRNAs for IGF‐I and IGF‐II and their cognate receptors are dramatically reduced in embryos cultured in SOM containing 125 mM NaCl, whereas only a mild reduction is observed in the relative amount of actin mRNA. In contrast, when freshly isolated morulae are cultured to the blastocyst stage in either of these 2 media, similar amounts of these mRNAs are observed. Last, high‐resolution, 2‐dimensional gel electrophoresis reveals significant changes in the pattern of protein synthesis when the embryos are cultured in SOM containing 125 mM NaCl. Results of these experiments suggest that culture of embryos in medium containing lower concentrations of NaCl that are normally present in various culture media results in higher rates of mRNA synthesis and greater mRNA stability. These changes in RNA synthesis may underlie, at least in part, the improved development in vitro that is fostered by SOM containing 85 mM NaCl. © 1994 Wiley‐Liss, Inc.
Assessment of sister chromatid exchange in mouse embryos produced by microsurgical fertilization
Authors: Hirayama Toshio , Quinn Patrick , Marrs Richard P.
Published: February 2005

The treatment of male factor‐related infertility has been approached with the advent of several methods for microsurgical fertilization, such as the partial dissection of the zona pellucida (PZD) and the injection of sperm into the perivitelline space (PVSI) of oocytes. These techniques are designed to increase sperm‐oolemma interaction by circumventing passage of the sperm through the zona pellucida. The present study was performed to evaluate the influence of PZD and PVSI on the in vitro development of mouse embryos by assessing the rate of sister chromatid exchange (SCE). SCE is considered to be a sensitive indirect indicator of DNA lesions due to various conditions. Oocytes were cultured in vitro after PZD or PVSI and then examined for SCE. There was no significant difference in SCE between control and treatment groups of embryos and the values were similar to those reported by Saito et al. (Fertil Steril 41:460–464, 1984). The rate of SCE was low during the first two mitotic cycles, then increased from cycle two to three before declining slightly between the 3rd and 4th cycles of cell division. These data demonstrate that the direct interaction of sperm and oocyte by PZD or PVSI did not have an adverse effect on the development of mouse embryos as assessed by the rate of SCE. © 1994 Wiley‐Liss, Inc.
In vitro incubation of golden (syrian) hamster ovarian oocytes and human sperm with a human oviduct specific glycoprotein
Authors: Reuter Laura M. , O'Day‐Bowman Mary B. , Mavrogianis Patricia A. , Fazleabas Asgerally T. , Verhage Harold G.
Published: February 2005

The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1‐D SDS‐PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre‐ and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct. © 1994 Wiley‐Liss, Inc.
Analysis of segregation in a human male reciprocal translocation carrier, t(1;11) (p36.3;q13.1) by two‐colour fluorescence in situ hybridization
Authors: Spriggs Elizabeth L. , Martin Renee H.
Published: February 2005

Using centromeric probes specific for chromosomes 1 and 11, 13,071 sperm nuclei from a male reciprocal translocation heterozygote, 46, XY, t(1;11) (p36.3;q13.1), were analyzed by fluorescence in situ hybridization (FISH). Decondensed sperm nuclei were simultaneously hybridized with DNA probes for chromosome 1 (pUC177) and chromosome 11 (D11Z1). Results were as follows: 1/11 (82.45%), 1/1/‐ (3.45%), ‐/11/11 (4.85%), 1/1/11 (1.20%), 1/11/11 (1.14%), 1/‐ (4.33%), ‐/11 (2.50%), 1/1/11/11 (0.06%), 1/1/1/‐ (0.02%). Because both the normal chromosome and its translocated derivative carry the same centromeric sequences, FISH cannot differentiate between sperm resulting from alternate segregation and those produced by adjacent I segregation. Using the same donor, comparable segregation patterns were obtained from sperm chromosome karyotypes (Spriggs et al., 1992: Hum Genet 88:447–452) and from MII spermatocytes (Goldman and Hulten, 1993: Cytogenet Cell Genet 63:16–23), demonstrating that selection is not a factor in the human sperm/hamster oocyte fusion technique or during meiosis. Although FISH does not provide the detailed information afforded by sperm karyotyping, it is a valuable technique for studying segregation patterns in translocation heterozygotes. © 1994 Wiley‐Liss, Inc.
Liposome‐mediated DNA transfer into chicken primordial germ cells in vivo
Authors: Watanabe M. , Naito M. , Sasaki E. , Sakurai Michiharu , Kuwana T. , Oishi T.
Published: February 2005

In embryogenesis, avian primordial germ cells (PGCs) circulate temporarily in the blood vessels at stages 10–15 (Hamburger and Hamilton, 1951), before reaching the gonads. In an attempt to transfer cloned genes into PGCs, liposome consisting of reporter plasmid DNA and N‐[1‐(2,3‐Dioleoyloxy)propyl]‐N, N, N‐trimethyl‐ammoniummethylsulfate was injected into the marginal veins of embryos at stages 11‐‐15. As reporter plasmids, pRSVZ and pAcZ harboring the Escherichia coli lacZ gene driven, respectively, by the Rous sarcoma virus (RSV) promoter and the chicken β‐actin gene promoter were used. First, 55 embryos were injected with liposome containing pRSVZ and stained for the bacterial β‐galactosidase activity 24 hr after injection. In all the embryos, cells positive for β‐galactosidase activity were observed among the blood cells, endothelial cells, and endocardium cells of the heart, suggesting that transfection took place within the circulatory system. Then, embryos were injected with liposome containing pRSVZ or pAcZ, and stained 2 or 3 d after injection. PGCs positive for β‐galactosidase activity were observed in the gonads in four out of 44 embryos injected with pRSVZ, and 29 out of 71 embryos injected with pAcZ, indicating that the plasmid DNA was transferred into PGCs developing normally. The average number of positive PGCs per embryo was 0.2 and 2.1, respectively, when pRSVZ and pAcZ were introduced. The difference in the number of positive PGCs detected after introduction of the two plasmids suggests that the actin promoter has a higher level of transcriptional activity in PGCs than does the RSV promoter. © 1994 Wiley‐Liss, Inc.
Proenkephalin transgenic mice: A short promoter confers high testis expression and reduced fertility
Authors: O'Hara Bruce F. , Donovan David M. , Lindberg Iris , Brannock Michael T. , Ricker Deborah D. , Moffatt Christopher A. , Klaunberg Brenda A. , Schindler Charles , Chang Thomas S. K. , Nelson Randy J. , Uhl George R.
Published: February 2005

The regulation and possible function of the preproenkephalin gene in testis were studied in vivo in transgenic mice containing: (1) bases −193 to +210 of the human proenkephalin gene and an additional one kilobase of 3' proenkephalin flanking sequence driving expression of bacterial chloramphenicol acetyltransferase (CAT), and (2) the same promoter and flanking sequences driving expression of a rat proenkephalin cDNA. Five lines of mice, designated HEC1–5, expressed the first construct and 10, HER1–10, the second. Each HEC male and many HER males showed dramatic expression of the transgene in the testis, although much lower expression was observed in the brain and other enkephalin‐producing tissues. High levels of expression in testis can thus be achieved with a very short promoter region and do not require intron A sequences previously considered necessary. Altered enkephalin expression may affect testicular function. One founder, HER8, displayed grossly abnormal testicular morphology and was completely infertile. A second founder, HER6, had low sperm motility. Two offspring from other lines also displayed subnormal fertility. These studies support a role for specific promoter sequences in testis expression and may further support a significant role for proenkephalin in testicular function. © 1994 Wiley‐Liss, Inc.
Isolation and partial purification of plasma membrane from porcine oocytes
Authors: Ash Kendall , Berger Trrish , Munn Robert J. , Horner Catherine M.
Published: February 2005

Egg plasma membrane (EPM) was isolated in comparatively large amounts from porcine slaughterhouse ovaries. Ovaries were minced, and the oocyte containing fluid was filtered to retrieve zona pellucidae–intact oocytes. The oocytes were homogenized and filtered again to remove zona pellucidae. The egg filtrate was subjected to differential centrifugation to remove membrane bound organelles and the remaining plasma membrane containing material was pelleted by ultracentrifugation. Plasma membranes were further separated from cellular material by sucrose density gradient centrifugation and were collected from portions of the gradient that correspond to the densities of plasma membrane. The purity of isolated plasma membranes was assessed by membrane marker enzyme analysis and transmission electron microscopy. Activities of the plasma membrane marker enzymes 5' nucleotidase and alkaline phosphatase increased from nondetectable levels in the egg filtrate to relatively high levels in the plasma membrane preparation. Marker enzymes for mitochondrial and lysosomal membranes fell from detectable levels in the egg filtrate to levels that were at the lower limits of the assays to detect in the final preparation. Evidence provided by binding of biotin‐labeled EPM to capacitated sperm suggests that the isolated EPM retains its biological activity. The procedure presented here represents a novel method of isolating procine egg plasma membranes for further study involving sperm–egg interaction. © 1994 Wiley‐Liss, Inc.
Antibody‐induced changes on rabbit sperm surface inhibit gamete interaction
Authors: Shaha Chandrima
Published: February 2005

Interaction of specific ligands with cell surface molecules may induce reorganization of surface components. A monoclonal antibody (B‐12) against sperm surface antigens of 40kDa size induced molecules on the plasma membrane overlying the acrosome of rabbit sperm to cluster in small aggregates at 0°C (patching). At an elevated temperature of 37°C these clusters of antigen antibody complexes collected into a large aggregate on one pole of the cell forming a cap (capping). This cap‐like structure showed a reduction in size over a period of time and eventually disappeared from the sperm surface. Inhibition of capping by sodium azide indicated that it is an energy‐dependent process. Patching of antigens did not require energy. Involvement of sperm head cytoskeleton in the process of capping was evident from potentiation of cap formation by cytoskeleton disrupting agents like cytochalasin B and D. Patching of antigen antibody complexes was not affected by either of the agents. The loss of antigen antibody complexes from sperm surface was mainly due to shedding of the complexes in the surrounding media. Sperm with patches of antigen antibody complexes did not adhere to oocytes. Sperm from the group where a majority of the sperm were denuded of the antigen antibody complexes also did not bind to oocytes. © 1994 Wiley‐Liss, Inc.
Purification and characterization of β‐glucuronidase from bull seminal plasma and its role in fertilization
Authors: Rethinaswamy Asokan , Yang Chul‐Hak , Srivastava Prakash N.
Published: February 2005

Bull seminal plasma contains high levels of β‐glucuronidase. The present study describes the isolation and characterization of β‐glucuronidase, and its role in fertilization. β‐glucuronidase was purified by ion exchange chromatography, saccharolactone‐agarose affinity chromatography, and gel filtration. The specific activity of the purified enzyme was 4,414 μmoles/mg protein/min. The purified enzyme showed a single band on 7.5% PAGE. On SDS‐PAGE, the enzyme appeared to consist of four identical subunits of Mr 75,000 each. The apparent Km and Vmax for β‐glucuronidase were 0.4 mM and 5.7 μmol/min using phenolpthalein mono‐β‐glucuronic acid as the substrate. β‐glucuronidase appeared to accelerate the cumulus dispersion in vitro. © 1994 Wiley‐Liss, Inc.
Characterization of chromatin‐condensing proteins during spermiogenesis in a neogastropod mollusc (Murex brandaris)
Authors: Càceres Carme , Ribes Enric , Muller Sylviane , Cornudella Lluís , Chiva Manel
Published: February 2005

During the process of chromatin cndensation in the spermiogenesis of the neogastropod mollusc Murex brandaris, the nuclear protein complement undergoes a complex series of changes. These changes lead to the appearance of three small protamines in the ripe sperm nuclei. We have characterized this system electrophoretically and at the compositions with antibodies elicited against a specific spermatozoan protamine. Our results indicate that the complex pattern of chromatin condensation during spermiogenesis in this species (M. brandaris) may be modulated by a series of post‐translational (and intranuclear) modifications of DNA‐interacting proteins, such as precursors to the sperm protamines. The amino acid composition of each sperm protamine is remarkably simple (lys + arg + gly ≥96 mol%). This system of spermiogenic/spermatozoal proteins in the neogastropod M. brandaris clearly differs from that in patellogastropods and archaeogastropods, and it may be helpful in understanding evolutionary changes in the chromatin condensation pattern during the spermiogenesis of gastropod molluscs. © 1994 Wiley‐Liss, Inc.
Epidermal growth factor enhances oocyte maturation in pigs
Authors: Ding Jianchi , Foxcroft George R.
Published: February 2005

Epidermal Growth Factor (EGF) has been reported to stimulate nuclear maturation in porcine oocytes (Sommer et al., 1992). The objective of this experiment was to test the effect of EGF alone or in combination with gonadotropins and follicular shell coculture, on cytoplasmic maturation of oocytes in vitro. A preliminary experiment tested the effective dose of EGF for stimulation of oocyte nuclear maturation in our culture system. Nuclear maturation rates (MII%) of 67.2 ± 4.6, 81.1 ± 8.6, and 80.7 ± 5.1 for oocyte complexes cultured in the presence of 0.1, 1, and 10 ng/ml EGF, respectively, were significantly higher (P < 0.05) than for oocytes cultured in the absence of EGF (18.1 ± 9.4%). In the main experiment a 2 × 2 × 2 factorial random complete block design was used to examine the effect of EGF (1.0 ng/ml) alone or in combination with gonadotropins and follicular shell coculture on cytoplasmic maturation. Cytoplasmic maturation was evaluated by the ability of oocytes to decondense sperm nuclei after sperm penetration. EGF alone did not stimulate cytoplasmic maturation in oocytes in vitro (P > 0.05). However, EGF showed a positive interaction (P < 0.05) with gonadotropin treatment on male pronuclear formation. We conclude that EGF alone can stimulate nuclear maturation in pig oocytes, and EGF can interact with gonadotropins to enhance cytoplasmic maturation. A high level of cytoplasmic maturation of in vitro matured pig oocytes could be achieved using a combination of EGF, gonadotropin, and follicular shell supplementation to the culture environment. © 1994 Wiley‐Liss, Inc.