bluuebunny/crossref_metadata_2025 huggingface dataset:
Function of FGF‐4 in limb development
Authors: Niswander Lee , Martin Gail , Tickle Cheryll , Vogel Astrid
Published: February 2005

The apical ectodermal ridge plays a central role in limb development through its interactions with the underlying mesenchyme. Removal of the AER results in cessation of limb outgrowth and leads to truncation of the limb along the proximo‐distal axis. The many functions attributed to the ridge include maintenance of the progress zone mesenchyme. Here, cells are stimulated to proliferate, are maintained in an undifferentiated state, and are assigned progressively more distal positional values as the limb grows. The AER also functions to maintain the activity of the polarizing region, a region of mesenchyme which is thought to provide the primary signal for patterning along the antero‐posterior axis.

We have begun to explore the function of fibroblast growth factor‐4 (FGF‐4) during limb development. FGF‐4, which encodes an efficiently secreted protein, is expressed in the AER. We have previously demonstrated that FGF‐4 protein can stimulate limb mesenchyme proliferation and can induce the expression of a downstream homeobox gene, Evx‐1 (homologue of the Drosophila even‐skipped gene), that is normally regulated by a signal from the AER. To determine to what extent FGF‐4 protein can substitute for the AER to allow normal limb outgrowth, we performed experiments on the developing chick limb in ovo. Remarkably, we find that after AER removal, the FGF‐4 protein can provide all the signals required for virtually normal outgrowth and patterning of the limb. Further studies indicate that proliferation of progress zone cells is not sufficient, and that an additional signal is produced by the posterior mesenchyme in response to FGF‐4 which enables progress zone cells to acquire progressively more distal fates. Thus FGF‐4 maintains progress zone activity through a combination of at least two signals—one that acts directly on progress zone cells to stimulate their proliferation, and one that acts indirectly by maintaining the production of patterning signal(s) by the posterior mesenchyme. We further show that failure of the posterior mesenchyme to produce this signal correlates with failure to maintain polarizing activity. This raises the possibility that the signal produced by the posterior mesenchyme and required for progressive proximo‐distal limb patterning is identical to the polarizing activity. Further experiments demonstrate that retinoic acid, which mimics the activity of the polarizing region, can supply this signal. In conclusion, the finding that a single growth factor can serve as both the direct and indirect signals required to maintain progress zone activity provides a simple mechanism for ensuring that growth and pattern formation are linked in the developing limb. © 1994 Wiley‐Liss, Inc.
Relationship between morphology and chromosomal constitution in human preimplantation embryo
Authors: Pellestor F. , Girardet A. , AndréO B. , Arnal F. , Humeau C.
Published: February 2005

In in vitro fertilization (IVF) procedures, morphologic embryo grading is the sole criteria for selection of embryos transferable in utero. Cytogenetic analysis of preimplantation embryos was performed to investigate the relationship between chromosomal status and morphologic quality of preimplantation eggs. Aneuploidy was the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism, and fragmentation were also found. Our results, pooled with data drawn from previous reports, demonstrated the prognostic value of the embryo grading system as a means for eliminating chromosomally abnormal embryos. In contrast, data suggested that some aspects of the IVF process might be responsible for the occurrence of these abnormalities. © 1994 Wiley‐Liss, Inc.
Pdha‐2: A model for studying transcriptional regulation in early spermatocytes
Authors: C. Iannello Rocco , C. Young Julia , Kola Ismail
Published: February 2005

Precise temporal and tissue‐specific expression of genes during spermatocyte differentiation is crucial for the formation of functional spermatozoa. However, the mechanisms that regulate gene expression during spermatogenesis are poorly understood. One testisspecific gene, Pdha‐2, is beginning to emerge as a potentially important model for the study of these events. This review focuses on our current understanding of the expression and regulation of Pdha‐2 during spermatogenesis. © 1994 Wiley‐Liss, Inc.
Frequency of X‐Y chromosome dissociation in mouse spermatocytes from interstrain crosses, recombinant inbred strains, and chimeras: Possible involvement of paternal genome imprinting
Authors: Krzanowska Halina , Wabik‐Śliz Barbara
Published: February 2005

The frequency of dissociation of the X‐Y chromosome bivalent in diakinesis‐metaphase I spermatocytes differs significantly between two inbred mouse strains, CBA (29%) and KE (7%), that were used to obtain reciprocal F1 hybrids, and to develop recombinant inbred (R1) strains. The level of X‐Y dissociation was significantly higher in (KExCBA)F1 hybrids sired by the CBA males (24%) than in reciprocal F1 hybrids (12%), revealing the inheritance after the father. Among 14 RI strains, nine were concordant with KE, one with CBA, and four had intermediate phenotype, significantly different from both progenitor strains. This shows that at least two genes are involved, and their possible linkage with agouti and Trf loci is suggested. The linkage with agouti was confirmed by testing additional 10 CBXE incipient RI strains. There was no significant difference in the level of X‐Y dissociation between EXCB RI strains derived from the original cross sired by the CBA males and CBXE RI strains derived from the reciprocal cross. The involvement of the Y chromosome‐linked factors was unlikely because it was found earlier (Krzanowska, 1989: Gamete Res 23:357–365) that two congenic strains, KE and KE.CBA, differing with respect to the source of the Y chromosome, had the same level of X‐Y dissociation. Thus, the difference obtained between reciprocal F1 hybrids is interpreted in terms of paternal genome imprinting imposed by CBA males and propagated only in the presence of some alleles derived from this strain.

Analysis of six KE ⟷ CBA‐T6 chimeras, among them three germ line chimeras, points to the conclusion that the tendency to low or high level of X‐Y chromosome dissociation is expressed rather autonomously by KE or CBA‐T6 spermatocytes (as recognized by a marker chromosome pair), respectively, and was not modified by the presence of somatic cells of the opposite strain. © 1994 Wiley‐Liss, Inc.
Use of PCR‐based methods for selection of integrated transgenes in preimplantation embryos
Authors: Wilmut I. , Colman A. , Garner I. , S. Carver A. , Cousens C. , T.O'Neill G.
Published: February 2005

The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one‐cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine β‐lactoglobulin‐human α1‐antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one‐cell mouse embryos. It has examined two PCR‐based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam‐methylation or the substitution of dTTP by dUTP. The dam‐sensitive DNA endonuclease Dpnl, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP‐DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid‐preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight‐cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos. © 1994 Wiley‐Liss, Inc.
Sex‐related growth rate differences in mouse preimplantation embryos in vivo and in vitro
Authors: Peippo Jaana , Bredbacka Peter
Published: February 2005

Sex‐related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley‐Liss, Inc.
Effect of estradiol and progesterone on c‐myc expression in the sea star testis and the seasonal regulation of spermatogenesis
Authors: Marsh Adam G. , Walker Charles W.
Published: February 2005

Analysis of the expression of cell cycle control genes provides a sensitive assay for mitotic and meiotic progression under experimental conditions. Transcription of the c‐myc proto‐oncogene and thymidine incorporation into DNA were both used to assay the mitogenic effects of estradiol and progesterone on the testicular germinal epithelium of the starfish Asterias vulgaris. Experimental in vitro studies during the annual spermatogenic cycle show that testes are not sensitive to mitotic stimulation by estradiol alone, whereas progesterone alone is mitotically inhibitory. The germinal epithelium can only be mitotically stimulated by estradiol after pretreatment with progesterone. The net increase in mitotic activity following a progesterone to estradiol transfer is dependent on the length of progesterone pretreatment. Overall, the seasonal pattern of testicular estradiol and progesterone levels evident in field populations of A. vulgaris (Hines et al., 1992a: Endocrinology 100:1468–1471) do not appear to be responsible for initiating spermatogonial mitosis at the onset of the annual spermatogenic cycle. Expression rates of the c‐myc proto‐oncogene are substantially more sensitive than thymidine incorporation in quantifying mitotic activity. Measuring proto‐oncogene expression provides an ideal experimental approach to investigations of the regulatory mechanisms that control growth and reproduction in marine organisms. © 1995 Wiley‐Liss, Inc.
Cloning and expression of a rat placental cDNA encoding a novel cathepsin L‐related protein
Authors: Conliffe Phyllis R. , Ogilvie Susan , Simmen Rosalia C. M. , Michel Frank J. , Saunders Paul , Shiverick Kathleen T.
Published: February 2005

Cathepsin L is a major lysosomal cysteine protease produced by mouse placenta and fibroblasts. This study characterizes a novel cathepsin L‐related mRNA expressed in rat placenta. Immunological and nucleotide screening of a rat placental library identified six positive clones, the largest, pCLRP‐9, being 924 base pairs in length. The combined sequences of all the clones contain an open reading frame of 711 nucleotides, a termination codon, a polyadenylation site, and 197 nucleotides of 3′ untranslated region, but lack the 5′ translation initiation codon. The pCLRP nucleotide sequence showed 60–64% identity to those of mouse, rat, and human cathepsin L. The deduced amino acid sequence of pCLRP codes for 237 amino acids, which align with the carboxy‐terminal sequence of cathepsin L and has the active site residues characteristic of the cysteine protease family. Northern blot analysis showed hybridization of pCLRP with a major mRNA transcript of 1.3 kilobases expressed in placenta, but not kidney or liver. In contrast, a cDNA for mouse pro‐cathepsin L hybridized with a transcript of 1.7 kilobases expressed in rat kidney, as well as placenta. During late gestation, steady‐state levels of rat placental pCLRP mRNA were highest on day 18, whereas those of mouse procathepsin L were greatest on day 20 of gestation. Antiserum to mouse cathepsin L cross‐reacted with four proteins of molecular weights 36,000 to 42,000 in rat placental culture medium, of which two were absent in the kidney. These data indicate that rat placenta expresses several species of cathepsin L‐type proteins, which may be involved in placental function and nutrient supply. © 1995 Wiley‐Liss, Inc.
Expression of the mouse testis‐determining gene Sry in male preimplantation embryos
Authors: Cao Qiu Ping , Gaudette Michelle F. , Robinson Douglas H. , Crain William R.
Published: February 2005

The testis‐determining factor in the mouse is encoded by the Sry gene on the Y chromosome. Transcripts of this gene have been shown previously to be present in the genital ridge at the beginning of gonadal differentiation (11.5 days post coitum) and in adult testis. In this study, RNA transcripts of the Sry gene are also detected in male blastocyst‐stage embryos (3.5 days post coitum) at approximately 40–100 copies per cell, long before overt sex differentiation. These results indicate that preimplantation mouse embryos have sexually dimorphic gene expression at least with respect to Sry transcripts. In addition, at least some of the Sry RNA transcripts in blastocysts are circular, as has been reported for Sry transcripts from adult testis. The appearance of Sry transcripts in blastocysts at this level raises the possibility that sex determination begins earlier during embryonic development than previously thought. © 1995 Wiley‐Liss, Inc.
Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis
Authors: Gu Wei , Kwon Yunhee , Oko Richard , Hermo Louis , Hecht Norman B.
Published: February 2005

RNA‐binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley‐Liss, Inc.
Activin βA subunit is expressed in bovine oviduct
Authors: Gandolfi F. , Modina S. , Brevini T. A. L. , Passoni L. , Petraglia F. , Lauria A. , Artini P.
Published: February 2005

It is evident that members of several growth factor families are actively involved in embryogenesis from its earliest phases. Several reports also indicate the oviduct as a possible source of growth factors, suggesting an active role of this organ in mammalian embryonic development. The aim of this study was to investigate the presence of activin/inhibin subunits in bovine oviduct since activin is a well‐characterised morphogen in amphibian development. The presence of transcripts for α. βA, and βB subunits was investigated by analysing oviduct epithelial cells mRNA with reverse transcription‐polymerase chain reaction (RT‐PCR). Moreover, antisera specific for the three subunits were used for the Western blot analysis of the proteins secreted by oviduct epithelial cells in vitro and for their immunohistochemical localisation in different oviductal regions. Oviduct epithelial cells expressed only the βA‐subunit gene. Immunoreactive material was present among in vitro secreted proteins, indicating that the transcript is translated into a polypeptide that has been localised in the epithelium of both the ampullary and isthmic tract of the organ. Consistent with these results, the antisera for the α and βB subunits did not recognise any specific antigen either among secreted proteins or in the sections. These results indicate that βA subunit gene is expressed in bovine oviduct epithelial cells, and the protein is secreted in vitro and can be found along the whole extension of the organ. In the absence of α or βB subunits, this suggests that activin A is present in bovine oviduct. Such a finding would be consistent with an embry‐otrophic activity of this organ, but definitive conclusions on the target tissue and the specific functions of oviductal activin require further studies. © 1995 Wiley‐Liss, Inc.
Cumulus cell function during bovine oocyte maturation, fertilization, and embryo development in vitro
Authors: Zhang Li , Jiang Shie , Wozniak Patricia J. , Yang Xiangzhong , Godke Robert A.
Published: February 2005

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm‐oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus‐free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures.

Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co‐culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley‐Liss, Inc.
Atrial natriuretic peptide: A chemoattractant of human spermatozoa by a guanylate cyclase‐dependent pathway
Authors: Anderson Robert A. , Feathergill Kenneth A. , Rawlins Richard G. , Mack Stephen R. , Zaneveld Lourens J. D.
Published: February 2005

Atrial natriuretic peptide (ANP), found in mammalian ovarian granulosa cells and oocytes (Kim et al., 1992, 1993), induces the human acrosome reaction (Anderson et al., 1994). The purpose of the present study was to determine whether ANP, as eggderived peptides from sea urchins, can act as a chemoattractant to human spermatozoa. Small lengths of capillary tubing that contained different concentrations of ANP were suspended over a suspension of washed spermatozoa. The number of spermatozoa that entered the tubing was determined. More than twice the number of spermatozoa moved into the tubing that contained a maximally effective concentration of ANP, as compared with tubing that contained only medium. The concentration of ANP that produced a half‐maximal effect was 0.7 nM. The effect was blocked by LY83583, an inhibitor of guanylate cyclase. ANP produced more than a twofold increase in the rate of cGMP formation, an effect that was blocked by LY83583. Human ANP (5–27), a fragment of the intact peptide, had no chemoattractant activity. These findings suggest that a specific sperm receptor exists for the chemoattractant activity of ANP that is associated with guanylate cyclase. The chemoattractant activity of ANP is independent of the presence of extracellular calcium ions and is independent of the action of ANP as a stimulus of the acrosome reaction. There is no association between the chemoattractant activity of follicular fluid and the follicular fluid concentration of ANP. These data suggest that factors besides ANP are responsible for the chemoattractant activity of follicular fluid. The present study adds to evidence that human ANP is a potent modulator of human sperm function. © 1995 Wiley‐Liss, Inc.
Role of glucose in mouse preimplantation embryo development
Authors: Martin Karen L. , Leese Henry J.
Published: February 2005

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one‐and two‐cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L‐lactate, in the presence and absence of 1 mM glucose (M16+G and M16‐G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16‐G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two‐cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two‐cell stage in M16‐G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two‐cell stage in M16‐G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One‐cell embryos cultured continuously in M16‐G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley‐Liss, Inc.
Detection of Y‐bearing porcine spermatozoa by in situ hybridization using digoxigenin‐labeled, porcine male‐specific DNA probe produced by polymerase chain reaction
Authors: Kawarasaki Tatsuo , Kohsaka Tetsuya , Sone Masaru , Yoshida Mitsutoshi , Bamba Kimio
Published: February 2005

This study was carried out to determine whether Y‐bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)‐labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig‐dUTP) was performed using a set of oligonucleotide primers (5′‐AAGTGGTCAGCGTGTCCATA‐3′ and 5′‐TTTCTCCTGTATCCTCCTGC‐3′) for 236 bp fragment of porcine male‐specific DNA sequence and 1.25 × 104 template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig‐labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig‐labelled DNA probe and alkaline phosphatase‐labeled anti‐Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig‐labelled DNA probe. The results indicated that in situ hybridization with porcine male‐specific DNA probe produced by PCR made possible the direct visualization of Y‐bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley‐Liss, Inc.
Synthesis and accumulation of p34cdc2 and cyclin B in mouse oocytes during acquisition of competence to resume meiosis
Authors: Chesnel Franck , Eppig John J.
Published: February 2005

This study tests the hypothesis 033 that growing murine oocytes, which are incompetent to resume meiosis, are deficient in their content of p34cdc2 and/or cyclin B, the two subunits of maturation promoting factor (MPF). Accumulation of the two MPF components occurred in an asynchronous manner in growing oocytes. Cyclin B content reached maximal levels in oocytes that were not yet competent to undergo germinal vesicle breakdown (GVB), the first obvious morphological manifestation of the resumption of meiosis. Thus, the amount of cyclin B is not the limiting factor rendering these growing oocytes incompetent to undergo GVB. In contrast, synthesis and accumulation of p34cdc2 increased during the period of oocyte growth in vivo when they became competent to undergo GVB. A similar increase in the amount of p34cdc2 also occurred in cultured granulosa cell‐free oocytes despite the lack of oocyte growth, but these cultured oocytes did not become GVB competent. Thus, the accumulation of p34cdc2 is probably necessary, but not sufficient, for mouse oocytes to become competent to undergo GVB. This accumulation occurs autonomously in oocytes independently of growth or of the participation of follicular somatic cells. © 1995 Wiley‐Liss, Inc.
Requirement for protein synthesis during the onset of meiosis in bovine oocytes and its involvement in the autocatalytic amplification of maturation‐promoting factor
Authors: Tatemoto Hideki , Horiuchi Toshitaka
Published: February 2005

The present study was carried out using the method of electrofusion, or treatment with okadaic acid (OA), to determine whether protein synthesis at the onset of culture was required for the meiotic resumption of bovine follicular oocytes. Germinal vesicle breakdown (GVBD) occurred in bovine oocytes at 6 hr after separation from their follicles in vitro. Following this, immature germinal vesicle (GV) oocytes, preincubated for 0,2,4, and 6 hr, were fused to mature oocytes. When immature oocytes, preincubated for 0 hr, were fused to mature oocytes and then cultured for 3 hr in basic medium, GVBD was observed in all fused cells, whereas in the case of cultivation in medium supplemented with the protein synthesis inhibitor (25 μg/ml cycloheximide; CX), 39% of the fused cells possessed an intact GV within their cytoplasm. In immature oocytes preincubated for 4 or 6 hr, however, this proportion was significantly reduced to 7% and 4%, respectively, without protein synthesis after fusion. In addition, the CX‐dependent block of GVBD could be overcome in only 13% of bovine follicular oocytes by the addition of 2 μM OA, although 51% of oocytes which synthesized the protein during the first 6 hr of culture induced GVBD in subsequent culture with CX plus OA. Thus, we conclude that the initiation of GVBD in bovine oocytes requires protein synthesized at the onset of meiosis, which is related to the autocatalytic amplification of the maturation‐promoting factor. © 1995 Wiley‐Liss, Inc.
Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes
Authors: Bos‐Mikich A. , Swann K. , Whittingham D. G.
Published: February 2005

We have examined the ability of the two parthenogenetic agents, strontium (Sr2+) and cycloheximide, to activate mouse oocytes. We demonstrate that Sr2+ and cycloheximide act synergistically to promote parthenogenetic activation up to the pronuclear stage in oocytes collected immediately after ovulation. These two agents appeared to act independently, since incubation in Sr2+ media triggered a series of intracellular Ca2+ rises without affecting protein synthesis and cycloheximide inhibited protein synthesis without causing any intracellular Ca2+ changes. In addition, cycloheximide did not alter the pattern of Ca2+ oscillations induced by Sr2+. In contrast, we show that another commonly used parthenogenetic activation treatment, the Ca2+ ionophore A23187, has dual effects. Exposure of oocytes to the Ca2+ ionophore, A 23187, in Ca2+‐ and Mg2+‐free medium leads to the activation of young oocytes. However, as well as generating a Ca2+ increase, the treatment of mouse oocytes with A23187 and Ca2+‐ and Mg2+‐free media led to a marked inhibition of protein synthesis. Our data show that parthenogenetic agents may have two important loci for activating mammalian oocytes and that the combined effect on Ca2+ release and protein synthesis is most effective. © 1995 Wiley‐Liss, Inc.
Cis‐regulatory elements and transcription factors involved in the regulation of the transforming growth factor‐β2 gene
Authors: Scholtz Beáta , Kelly David , Rizzino Angie
Published: February 2005

Embryonal carcinoma (EC) cells and embryonic stem (ES) cells provide useful model systems for studying differentiation during early mammalian development. Previous studies have demonstrated that differentiation of two restricted mouse EC cell lines is accompanied by activation of the TGF‐β2 gene. Moreover, one negative and two positive regulatory regions upstream of the transcription start site were identified, which appear to play key roles in the transcriptional regulation of the human TGF‐β2 gene. In this report, we demonstrate that the same three regulatory regions strongly influence the activity of the TGF‐β2 promoter in differentiated cells derived from the multipotent human EC cell line, NT2/D1, and from the murine totipotent ES cell line, CCE. We also determined that the same three regions are active in the regulation of the TGF‐β2 gene in the murine parietal endoderm‐like cell line, PYS‐2. However, an additional negative regulatory region appears to contribute to the regulation of the TGF‐β2 gene in PYS‐2 cells. Last, mutation of a CRE/ATF element located just upstream of the transcription start site of the TGF‐β2 gene reduces significantly the activity of the TGF‐β2 promoter in the differentiated cells. However, in contrast to our previous findings, our gel mobility shift analyses demonstrate that this CRE/ATF element is bound by similar proteins in nuclear extracts prepared from undifferentiated and differentiated mouse EC cells as well as from undifferentiated human EC cells. Taken together, our findings suggest strongly that four regulatory regions, including a critical CRE/ATF element, help control the expression of the TGF‐β2 gene during early mammalian development. © 1995 Wiley‐Liss, Inc.
Effect of microinjection time during postfertilization S‐phase on bovine embryonic development
Authors: Gagné Marc , Pothier François , Sirard Marc‐André
Published: February 2005

Microinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken β‐actin promoter‐lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H‐thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21–22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9–18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin‐treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P<0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (>16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin‐ and cycloheximide‐treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P<0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage. © 1995 Wiley‐Liss, Inc.
Endocytosis in mouse blastocysts: Characterization and quantification of the fluid phase component
Authors: Dunglison Greta F. , Kaye Peter L.
Published: February 2005

Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, 3H‐dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy‐requiring process, with only 20% accountable by diffusion as indicated by analysis at 4°C. Insulin stimulated uptake of 3H‐dextran by 30% (P<0.05) over the first hr. The rate of uptake then decreased in both control and insulin‐treated blastocysts. However, by 2 hr, insulin‐treated blastocysts contained 38% more 3H‐dextran (38%; P<0.01) than control blastocysts. Incubation of blastocysts in protein‐free medium increased 3H‐dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 ± 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/I and 10 g BSA/I, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid‐phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient‐containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells. An analysis of the rate of protein delivery in both adsorbed and dissolved phases is presented, which reveals the potential for significant contributions of both phases of endocytosis to blastocyst metabolism in vivo. © 1995 Wiley‐Liss, Inc.
An isoform of ferritin as a component of protein yolk platelets in Schistosoma mansoni
Authors: Schüßler Peter , Pötters Elke , Winnen Ralf , Bottke Werner , Kunz Werner
Published: February 2005

Schistosoma mansoni possesses two isoforms of the iron storage protein ferritin, Fer1 and Fer2. At the mRNA level as well as at the protein level, Fer1 is much more abundant than Fer2; females contain an about 15‐fold excess of Fer1 compared with males. In contrast, nearly equal amounts of Fer2 occur in both sexes. By electron microscopy we identified ferritin as a component of electron dense membrane‐bound bodies in cells of the vitellarium. The mode of formation of these inclusions (as inferred from electron microscopy) and the abundance of phospholipid multilayered membranes suggest that these bodies are of a lysosomal nature. Here we interpret these ferritin‐containing inclusions as protein yolk platelets. To date, most of the literature does not contain any hints of the existence of protein yolk in trematodes. The possible function of ferritin in embryonic development is discussed. © 1995 Wiley‐Liss, Inc.
Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction
Authors: Dandekar Pramila , Mate Karen E. , Talbot P.
Published: February 2005

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule‐reacted oocytes from a marsupial, the grey short‐tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat‐tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short‐tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley‐Liss, Inc.
Chromatin condensation activity and cortical activity during the first three cell cycles of a mouse embryo
Authors: Ciemerych Maria A.
Published: February 2005

One‐cell parthenogenetic haploid embryos and blastomeres of the 2‐ and 4‐cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one‐cell embryos and of blastomeres.

The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2‐ and 4‐cell embryos did not exhibit any cortical activity.

In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2‐cell and 4‐cell stage blastomeres did not reveal condensation activity. © 1995 Wiley‐Liss, Inc.
Different transmission rates of herpesvirus thymidine kinase reporter transgenes from founder male parents and male parents of subsequent generations
Authors: Ellison Aaron R. , Wallace Helen , Al‐Shawi Raya , Bishop John O.
Published: February 2005

Previously we demonstrated that lines of transgenic mice carrying the herpes simplex type 1 virus thymidine kinase (HSV1‐tk) reporter gene are malesterile. Ectopic transcription of the HSV1‐tk reporter in the testis was initiated downstream of the normal translation initiation codon and truncated proteins consistent with translational initiation at the second and third ATG codons were synthesized. Here we describe the effects on fertility (1) of converting the second and third ATG codons of the HSV1‐tk reporter to CTG codons and (2) of utilizing the HSV type 2 thymidine kinase (HSV2‐tk) reporter gene, in which the second ATG codon is located downstream of the ATP‐binding pocket of the enzyme. Both reporters were coupled to the bovine thyroglobulin promoter (bTG‐tk1α and bTG‐tk2 transgenes). The level of ectopic expression of these transgenes in the testis, relative to expression in the thyroid, was one to two orders of magnitude less than that of bTG‐tk1.

Sixty percent of male founders carrying the bTG‐tk1α and bTG‐tk2 transgenes were fertile but did not transmit the transgene. In contrast, most males from subsequent generations were fertile and transmitted the transgenes at the expected frequency. This difference between founder males and male descendants is also observed with certain constructs in which the HSV1‐tk reporter is coupled to other promoters. We attribute the effect to mosaicism among male founders, leading to competition between transgenic and nontransgenic spermatozoa and/or spermatogenic precursor cells and resulting in a lack of fertilization by transgenic sperm that would successfully fertilize eggs in the absence of competition. © 1995 Wiley‐Liss, Inc.
Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth
Authors: Yelian Frank D. , Yang Yan , Hirata Janie D. , Schultz Jeffery F. , Armant D. Randall
Published: February 2005

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum‐free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin‐binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell‐binding domain (FN‐120) and the Arg‐Gly‐Asp (RGD) recognition sequence. A 40 kD COOH‐terminal chymotryptic fragment of fibronectin containing both a heparin‐binding region and an alternate (non‐RGD) cell‐binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin‐binding domain, including the CS1 alternate cell‐binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val‐Thr‐Gly‐Arg‐Gly‐Asp‐Ser‐Pro‐Ala‐Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin‐mediated outgrowth. These findings demonstrate the key role of the central cell‐binding domain of fibronectin in trophoblast adhesion, and suggest four RGD‐binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley‐Liss, Inc.
Localization of intracellular calcium during the acrosome reaction in ram spermatozoa
Authors: Watson P. F. , Plummer J. M. , Jones P. S. , Bredl J. C. S.
Published: February 2005

Calcium was identified by a pyroantimonate‐osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time.

Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley‐Liss, Inc.