bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro
Authors: S. Melendrez Carman , Meizel Stanley , Berger Trish
Published: February 2005

It has been previously shown that progesterone can initiate the acrosome reaction (AR) of capacitated human and hamster sperm in vivo. We report here that progesterone can initiate a morphologically normal AR in porcine sperm that have undergone capacitation in a Hepes‐buffered medium in vitro. In addition, we have compared the abilities of progesterone and heat‐solubilized porcine zona pellucida (zona) to initiate the porcine sperm AR. Capacitated porcine sperm were treated with 1 m̈g/ml progesterone, 150 m̈g/ml porcine zona, or solvent control for 10 min. After treatment, sperm were incubated with the supravital dye Hoechst 33258, fixed and the acrosomal status determined in the previously viable sperm by fluorescence microscopy with fluorescein isothiocyanate‐labeled Pisum sativum agglutini (FITC‐PSA). There was no significant difference between the percentage of AR initiated by zona compared to that initiated by progesterone. In order to determine whether there was a synergistic interaction between the two AR initiators, both were added simultaneously to capacitated porcine sperm at optimal (1 m̈g/ml progesterone, 150 m̈g/ml zona) and suboptimal (75 ng/ml progesterone and 75 m̈g/ml zona) concentrations. Simultaneous addition of the two AR‐initiators at the two concentrations stimulated an additive AR‐initating response, rather than a synergistic one. Several possible explanations for the additive results are discussed. © 1994 Wiley‐Liss, Inc.
Translational regulation of the gradual increase in histone H1 kinase activity in maturing mouse oocytes
Authors: Hampl Aleš , Eppig John J.
Published: February 2005

In maturing mouse oocytes, p34cdc2‐associated histone H1 kinase activity gradually increases until it reaches its maximum at metaphase I (Choi et al., 1991: Development 113:789–795). In this study, treatment of oocytes with cycloheximide resulted in a failure to increase the level of histone H1 activity above that detected at approximately the time of germinal vesicle breakdown (GVB), which is ∼20‐30% of the level normally achieved at metaphase I. Cyclin B was detected in GV‐stage oocytes, but there was a 2–2.5‐fold increase in the amount of cyclin B in maturing oocytes from GV‐stage to metaphase I and a burst of cyclin B synthesis during the first 3 hr of maturation. Okadaic acid‐treatment of mouse oocytes did not accelerate activation of histone H1 kinase but rather arrested its activity at the same level observed in cycloheximide‐treated oocytes. Thus the components of the p34cdc2 kinase activating system in mouse oocytes are apparently not present in GV‐stage oocytes in an amount or configuration that would allow maximum kinase activation when meiosis is reinitiated by okadaic acid. Importantly, okadaic acid‐treatment dramatically inhibited protein synthesis. Therefore, the inhibition of protein synthesis by okadaic acid probably abrogates the possibility of de novo synthesis of the regulators of p34cdc2 kinase required to drive its activity to the maximum level normally achieved by metaphase I. It is concluded that there is a critical point in driving the continued activation of histone H1 kinase that occurs at approximately the time of GVB. Progression beyond this point requires de novo protein synthesis. Since newly synthesized cyclin B is immediately complexed with the p34cdc2 kinase in maturing mouse oocytes, cyclin B is a candidate for one of the proteins whose synthesis is required to drive the continued increase in histone H1 kinase activity after the time of GVB. © 1995 Wiley‐Liss, Inc.
Mitochondrial biogenesis in early mouse embryos: Expression of the mRNAs for subunits IV, Vb, and VIIc of cytochrome c oxidase and subunit 9 (P1) of H+‐ATP synthase
Authors: Taylor Kent D. , Pikó Lajos
Published: February 2005

The mouse egg contains about 90,000 mitochondria which undergo a buildup of mitochondrial cristae and increase in respiratory activity during cleavage. The mitochondrial DNA does not replicate during preimplantation development but is transcribed actively from the two‐cell stage onward (Pikó and Taylor, 1987: Dev Biol 123:364–374). To gain further insight into mitochondrial biogenesis, we have now determined the steady state amounts of the mRNAs for the cytochrome c oxidase (COX) subunits IV, Vb and VIIc and the H+‐ATPase subunit 9(P1) (all encoded by nuclear genes) in slot hybridization experiments of total RNA from oocytes and early embryos. All four mRNAs showed a similar developmental pattern of prevalence, characterized by a steady decline in mRNA copy numbers from the late growth‐phase oocyte through the two‐cell embryo, and an about 30‐fold rise during cleavage through the blastocyst stage. However, the ATPase subunit 9 (P1) mRNA was about three times more prevalent in cleavage‐stage embryos than the COX mRNAs. A similar pattern was obtained previously for the mitochondrial‐encoded COX I and II mRNAs, but the latter accumulate at a 30–50‐fold excess over the nuclear‐encoded COX subunit mRNAs during the cleavage stages. The results suggest a coordinated activation and transcription of the mitochondrial and nuclear genes for the components of the respiratory apparatus beginning with the two‐cell stage. It is estimated that new respiratory chains are produced at a rate of 50–100 chains hr‐1/mitochondrion in the early blastocyst, accounting for 3.5‐7% of the total protein synthetic activity at this stage. This rapid buildup of the mitochondrial oxidative phosphorylation system appears to be mostly preparatory for postimplantation development. © 1995 Wiley‐Liss, Inc.
Expression of the rabbit sperm protein Sp17 in cos cells and interaction of recombinant Sp17 with the rabbit zona pellucida
Authors: O'rand Michael G. , Richardson Richard T. , Yamasaki Noriyuki
Published: February 2005

This study extends our analysis of rabbit recombinant Sp17 (rSp17) by examining whether rSp17 synthesized in transfected COS cells will show a particular localization within the cell and whether the COS cell will bind with zona pellucida. We show, using the cross‐linking, reagent DSS that rSp17 can bind to rabbit zona glycoprotein R45 or R55. © 1995 Wiley‐Liss, Inc.
Effects of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro fertilization, development, and chromosomal abnormalities
Authors: Bouquet M. , Selva J. , Auroux M.
Published: February 2005

In a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen‐thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing‐thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0°C had no effect. DMSO, used at 0°C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing‐thawing process.
Regulation of the transforming growth factor‐β2 gene promoter in embryonal carcinoma cells and their differentiated cells: Differential utilization of transcription factors
Authors: Kelly David , Scholtz Beáta , Orten Dana Jo , Hinrichs Steven H. , Rizzino Angie
Published: February 2005

Previous studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF‐β2 gene and identified a CRE/ATF‐like motif in the TGF‐β2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF‐like motif. To test this possibility, we performed gel mobility shift analysis using double‐stranded oligodeoxynucleotides containing the TGF‐β2 CRE/ATF‐like motif and nuclear extracts prepared from F9 EC cells and F9‐differentiated cells. We determined that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF‐1 specific antibody. This observation is consistent with our Western immunoblot analysis that detects ATF‐1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF‐like motif. Together, our studies identify a likely role for the CRE/ATF‐like motif in the regulation of TGF‐β2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed. © 1995 Wiley‐Liss, Inc.
Identification and localization of integrin subunits in oocytes and eggs of the mouse
Authors: Evans Janice P. , Schultz Richard M. , Kopf Gregory S.
Published: February 2005

Results of a recent study have implicated egg integrins in sperm binding to the egg plasma membrane (Blobel et al., 1991: Nature 356:248–252). In this report, immunoprecipitation was used to identify, and confocal immunofluorescence microscopy was used to localize, several different integrin subunits in mouse eggs. Antibodies to α2, α5, αv, and β1 subunits, as well as antibodies to the fibronectin receptor (FNR; α5β1 and/or α3β1) and vitronectin receptor (VNR; αvβ3 and/or αvβ5), detect polypeptides of the appropriate molecular weights following immunoprecipitation. β1 is localized preferentially to either the microvillar or amicrovillar membrane/cortical regions of eggs, and these asymmetric localizations depend on the antibody used. Proteins recognized by anti‐FNR antibodies are localized preferentially to the amicrovillar membrane/cortical region. Germinal vesicle‐intact oocytes display a symmetric plasma membrane distribution using β and FNR antibodies, and the asymmetric distribution develops as a consequence of oocyte maturation and is clearly observed by metaphase I. In contrast to the membrane localization of these integrin subunits, α2, α5, and VNR are predominantly localized in the cytoplasm of both oocytes and eggs. In the oocyte, each of these integrin subunits is uniformly distributed throughout the cytoplasm. Oocyte maturation is associated with a redistribution of α5 and VNR, leading to an asymmetric cytoplasmic distribution with an increased localization towards the spindle. αv, which is localized in the plasma membrane/cortex of both oocytes and eggs, does not show such a change during oocyte maturation. Results of these experiments are discussed in the context of a role for integrins in mediating sperm plasma membrane‐egg plasma membrane interactions leading to egg activation. © 1995 Wiley‐Liss, Inc.
Aneuploidy in late‐step spermatids of mice detected by two‐chromosome fluorescence in situ hybridization
Authors: Wyrobek A. , Lowe X. , Pinkel D. , Bishop J.
Published: February 2005

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late‐step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick‐translated with biotin‐ or digoxigenin‐labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late‐step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in ∼ 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late‐step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, ∼ 3/10,000 spermatids had fluorescence phenotypes indicative of X‐X or 8–8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of “bridging biomarkers” between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin. © 1995 Wiley‐Liss, Inc.
Factors involved in nuclear reprogramming during early development in the rabbit
Authors: Pinto‐Correia Clara , Long Charles R. , Chang Thomas , Robl James M.
Published: February 2005

Mechanisms of nuclear reprogramming and assessment of potential malfunctions that could be deleterious for development were evaluated in rabbit zygotes, parthenotes, and nuclear transfer embryos by analysis of DNA replication, nucleolar fibrillarin label, and localization of nuclear material reactive to the MPM‐2 antibody. Nuclear transfer embryos were derived from G1/early S‐phase donor nuclei and MII oocytes. In nuclear transfer embryos, DNA rerelication was likely to have occurred because label was incorporated, possibly in the centromeric regions of the chromosomes, prior to premature chromosome condensation and again following pronuclear formation. In parthenotes, DNA replication began very late in the cell cycle, which may be due to deficiencies in the artificial activation stimulus. The presence of fibrillarin label in the nucleolus was used as an indication of nucleolar transcriptional activity. Fibrillarin label was absent in embryos of all types up to the 16‐32‐cell stage. Although fibrillarin reappeared in nuclear transfer and parthenote embryos at the appropriate stage, not all blastomeres showed label indicating impaired development in these embryos. Labelling of phosphorylated epitopes by MPM‐2 antibody showed a change in pattern of labelling during early development. Early cleavage stage embryos did not exhibit labelling over the spindle poles as did blastomeres from 32‐cell embryos and tissue culture cells. All cell types exhibited labelling during interphase as dots located primarily over the nucleus in blastomeres from 32‐cell embryos and in tissue culture cells, together with cytoplasmic label in embryos at early cleavage stages. Nuclear transplant embryos had a normal pattern of MPM‐2 label. In contrast, the appearance of MPM‐2 label in parthenotes depended on the type of calcium stimulation. These results demonstrate defects in DNA synthesis, nucleolar activity, and specific phosphorylation events, likely resulting from an improper activation stimulus and chromosome condensation in the transplanted nucleus. © 1995 Wiley‐Liss, Inc.
Localization of fibrillarin and nucleolin in nucleoli of mouse preimplantation embryos
Authors: Baran V. , Veselá J. , Rehák P. , Koppel J. , Fléchon J. E.
Published: February 2005

The localization of fibrillarin and nucleolin in the nuclei of mouse two‐cell, four‐cell, and eight‐cell embryos has been studied using immunofluorescent staining with specific antibodies. In all of these cleavage stages, both antigens were associated exclusively with the peripheral region of the nucleolus precursor bodies (NPBs). The original speckled fluorescent staining pattern in the early two‐cell stage was progressively changed into a continuous fluorescent‐positive layer localized in the cortex of the NPBs in the four‐cell embryos. The compact central area of NPBs was never stained. Both proteins were colocalized in the same substructures of developing nucleoli. In order to analyze the interaction of chromatin, with NPBs, DNA structures were specifically immunolbelled. At the time of resumption of nucleolar transcription (in the two‐cell mouse embryo), DNA was detected at the periphery of, but not penetrating into, NPBs. Our results confirm the view that the cortical region of NPBs could represent a nucleolonemal area involved in the resumption of nucleolar transcription in the early mouse embryo. © 1995 Wiley‐Liss, Inc.
Effects of stimulators of protein kinases A and C and modulators of phosphorylation on plasminogen activator activity in porcine oocyte‐cumulus cell complexes during in vitro maturation
Authors: Kim Nam Hyung , Menino Alfred R.
Published: February 2005

Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6‐dimethylami‐nopurine (6‐DMAP), and okadaic acid (OA) on plasminogen activator (PA) activity in porcine oocyte‐cumulus cell complexes (POCC) in vitro were determined. Cumulus cell‐enclosed oocytes were collected from 1–4 mm antral follicles and cultured in TCM‐199 with 0.3% polyvinyl‐pyrrolidone for 48 hr PA activities in POCC were quantified using SDS‐PAGE, casein‐agar zymography, and densitometry. Two plasminogen‐dependent lytic zones (93–96 kD and 71–79 kD) were observed in POCC. Addition of amilorde to the zymograph, a competitive inhibitor of urokinase‐type PA, failed to reduce activities in either zone, suggesting that the 71–79 kD band is a tissue‐type PA (tPA) and the 93–96 kD band is possibly a tPA‐inhibitor complex. Changes in PA activity due to the various treatments were expressed relative to the PA activity in 40 POCC. Increasing dbcAMP increased PA (P <0.05) activity in dose‐dependent fashion, whereas 6‐DMAP and 10 and 100ng/ml PMA inhibited (P <0.05) PA activity. PA activity increased (P <0.05) in POCC treated with up to 25 nM OA; however, activity decreased (P <0.05) at concentrations >75 nM. Treatment with 25 nM OA also induced the expression of an amiloride‐sensitive PA (49–52 kD). Germinal vesicle breakdown and progression to metaphase II were inhibited (P <0.05) by 2.5 mM dbcAMP and 2 mM 6‐DMAP, whereas 100 ng/ml PMA and 25 nM OA inhibited (P <0.05) only progression to metaphase II. These data suggest that PA production by POCC is influenced by protein kinases A and C and kinase inhibitors during oocyte maturation. Inhibition of intracellular phosphatases also induced novel PA production. © 1995 Wiley‐Liss, Inc.
Efficient production of transgenic cattle by retroviral infection of early embryos
Authors: Haskell R. E. , Bowen R. A.
Published: February 2005

Production of transgenic cattle by microinjection of DNA has been difficult and costly. To explore an alternative method, one‐ to four‐cell bovine embryos were exposed to a replication‐defective retrovirus by microinjection of retrovirus producer cells into the perivitelline space. Embryos were cultured in vitro for 3–4 days, then transferred to recipient cows for further development. Thirteen of 22 embryos recovered at 15 days gestation and each of four fetuses recovered at 90 days gestation were transgenic. Fetuses harbored between 2 and 12 pro‐viruses, and within each fetus, identical patterns of integration were observed in seven tissues tested. Estimates of the number of proviruses per cell suggested that in three of the four fetuses, most, and possibly all, cells were transgenic. This technique should facilitate application of transgenic technology to cattle and other agriculturally important species. © 1995 Wiley‐Liss, Inc.
Epidermal growth factor and its receptor gene expression and peptide localization in porcine ovarian follicles
Authors: Singh Balwant , Rutledge Jean M. , Armstrong David T.
Published: February 2005

The present study was undertaken to determine the expression of genes for epidermal growth factor (EGF) and its receptor (EGF‐R) in various components of medium‐sized porcine ovarian follicles by reverse transcription‐polymerase chain reaction (RT‐PCR), and to localize their peptides during folliculogenesis by immunocytochemistry. A strong band for EGF mRNA transcript was detected in the oocyte, whereas the signal in cumulus, granulosa, and theca cells was very weak but detectable. In contrast, a very strong EGF‐R mRNA signal was observed in cumulus, granulosa, and theca cells, whereas the signal in the oocyte was very weak. EGF peptide was localized in the oocyte, cumulus, and granulosa cells of all stages of follicle. In the oocyte, the intensity of immunostaining was more pronounced in primordial and primary follicles, compared to antral follicles. In large antral follicles, immuno‐staining was pronounced in granulosa cells, whereas theca cells showed little or no detectable staining for EGF. EGF staining was also observed in the cumulus and granulosa cells of follicles undergoing atresia. EGF‐R immunostaining was observed in the oocytes of primordial and primary follicles, and in cumulus, granulosa, and theca cells of all stages of follicle, including atretic follicles. In large antral follicles, the intensity of immunostaining was more pronounced in theca cells than in granulosa cells, and the oocyte showed little or no detectable staining. No immunostaining was observed when the primary antibody was replaced with preimmune serum (EGF), or preabsorbed with the control peptide (EGF‐R), confirming the specificity of the staining procedures. These results suggest a local follicular production of EGF and its receptor in the porcine ovary, and thus a role for EGF of follicular origin in the regulation of follicular development in autocrine/paracrine fashion. © 1995 Wiley‐Liss, Inc.
Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts
Authors: Van Stekelenburg‐Hamers Annelies E. P. , Van Achterberg Tanja A. E. , Rebel Heggert G. , Fléchon Jacques E. , Campbell Keith H. S. , Weima Sjerp M. , Mummery Christine L.
Published: February 2005

Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal‐stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight‐cell block. © 1995 Wiley‐Liss, Inc.
Mitogen‐activated protein (MAP) kinase during the acquisition of meiotic competence by growing oocytes of the mouse
Authors: Harrouk Wafa , Clarke Hugh J.
Published: February 2005

During the growth phase of oogenesis, oocytes acquire the ability to undergo meiotic maturation. Although the molecular basis of this meiotic competence is unknown, specific differences in microtubular organization exist between incompetent and competent mammalian oocytes. Mitogen‐activated protein (MAP) kinase has been implicated in microtubular regulation and is present in fully grown competent oocytes of mice, suggesting a possible role for this protein in the acquisition of meiotic competence. We report that the MAP kinase species, p42ERK2 and p44ERK1, were detectable by immunoblotting in incompetent oocytes at the early stages of oocyte growth and throughout subsequent growth and acquisition of competence. In partially competent oocytes, which can enter metaphase but cannot complete the first meiotic division, both p42ERK2 and p44ERK1 became phosphorylated, as judged by retarded electrophoretic mobility, and a morphologically normal spindle was assembled. In incompetent oocytes, which cannot enter metaphase, p42ERK2 and p44ERK1 remained nonphosphorylated. When these oocytes were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, a portion of them entered metaphase and the slow‐migrating phosphorylated forms of p42ERK2 and p44ERK1 were observed. These phosphorylated forms appeared more rapidly, relative to the time of entry into metaphase, than during maturation of fully competent oocytes. The remaining incompetent oocytes, which did not enter metaphase during okadaic acid treatment, also did not generate slow‐migrating p42ERK2 and p44ERK1. These results suggest that the acquisition of meiotic competence during oocyte growth is not linked to the de novo appearance of p42ERK2 or p44ERK1, that the failure of partially competent oocytes to complete meiosis I reflects a defect acting downstream or independently of MAP kinase phosphorylation, and that the ability of meiotically incompetent oocytes to generate phosphorylated forms of p42ERK2 and p44ERK1 in response to okadaic acid is linked to the ability to enter metaphase. © 1995 Wiley‐Liss, Inc.
Differential effects of protein synthesis inhibitors on porcine oocyte activation
Authors: Nussbaum Diana J. , Prather Randall S.
Published: February 2005

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L‐[35S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D‐SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25‐kDa polypeptide to a 22‐kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley‐Liss, Inc.
Possible role of variant RNA transcripts in the regulation of epidermal growth factor receptor expression in human placenta
Authors: Ilekis John V. , Stark Benjamin C. , Scoccia Bert
Published: February 2005

Epidermal growth factor receptor (EGFR) plays an important role in growth and differentiation. The human placenta expresses high levels of the receptor. In the placenta, as in many other human tissues, EGFR is encoded by two RNA transcripts of 5.8 kb and 10.5 kb. The placenta also expresses a putative truncated EGFR transcript of 1.8 kb, which encodes only the ligand binding domain of the receptor. The etiology and role of these variant EGFR transcripts is unknown. Using the human placenta as a model to study this area, we report (1) the relationships among these transcripts suggest that the induction of alternate pathways of EGFR RNA processing is involved in their etiologies; (2) the 10.5 kb transcript may be the principal transcript involved in determining the level of the protein receptor; and (3) the isolation of a soluble protein with characteristics consistent with a translational product corresponding to the 1.8 kb transcript, which may act in regulating the activity of EGFR. Together these results suggest that alternate processing of EGFR RNA into variant transcripts may represent a novel mechanism involved in the regulation of the receptor protein. © 1995 Wiley‐Liss, Inc.
Nonoverlapping expression of Cx43 and Cx26 in the mouse placenta and decidua: A pattern of gap junction gene expression differing from that in the rat
Authors: Pauken Christine M. , Lo Cecilia W.
Published: February 2005

We characterized the expression of two gap junction genes (Cx26 and Cx43) in the mouse decidua and placenta. In the decidua, in situ hybridization analysis and immunostaining studies revealed a high level of Cx43 expression. In contrast, Cx26 expression was not detected. Analysis of the placenta revealed that both Cx43 and Cx26 transcripts are expressed, but in nonoverlapping cell populations. Cx26 transcripts were observed only in the labyrinthine trophoblast layer of the placenta, a tissue of ectoplacental cone derivation. In contrast, no Cx43 transcripts were found in the placenta proper, but only in the maternally derived decidual cap covering the placenta. These results, in conjunction with previous observations in the mouse and rat, indicate that there may be species‐specific differences in the pattern of Cx43/Cx26 expression in the placenta and decidua. © 1995 Wiley‐Liss, Inc.
Action of metallic ions on the precocious development by rabbit sperm of motion patterns that are characteristic of hyperactivated motility
Authors: Young R. J. , Bodt B. A. , Heitkamp D. H.
Published: February 2005

An earlier study demonstrated that rabbit sperm incubated for 16 hr under capacitation conditions acquire motility patterns identical to those seen in rabbit sperm capacitated in vivo. We now show that similar motion patterns develop after 0.5 hr incubation in a Trisbuffered medium, medium M. Development and decline of the motion patterns occurred in three phases each recognized by the character of the biphasic motion patterns. Hyperactivated sperm were objectively identified and quantified by a previously developed computer‐directed model. The percentage of motile sperm that acquired hyperactivated motility and the period they remained in this state varied among sperm from different rabbits. The decline in hyperactivated motility was paralleled by a decrease in the average sperm curvilinear velocity (VCL) and average amplitude of lateral head displacement (AALH), but was not accompanied by a concomitant decrease in percentage of motile sperm. Pb2+ and Cd2+, at concentrations that did not inhibit motility, prevented development of hyperactivated motility. Inhibition of hyperactivated motility by Pb2+ was time‐ and concentration‐dependent; the average percentage of hyperactivated sperm decreased from ∼ 30% to<5% (n = 5) in 1 hr at a Pb2+ concentration of 25 μM. Cd2+ inhibition of hyperactivation was dependent only on concentration of the cation. At a concentration of 100 μM, the decrease in the percent of hyperactivated sperm was ∼ 50% (n = 3). Hg2+, Zn2+, and Cr6+ at sublethal concentrations had no effect on hyperactivated motility development. These results suggest that Pb2+ and Cd2+, by virtue of their ability to prevent the wide curvature flagella beating that is characteristic of hyperactivation, can compromise fertilization at concentrations that do not inhibit sperm motility and act as a reproductive toxicant at a level other than spermatogenesis. © 1995 Wiley‐Liss, Inc.
Sperm‐induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery
Authors: Tesarik Jan , Sousa Mario , Mendoza Carmen
Published: February 2005

Temporal and spatial characteristics of explosive periodic increases (spikes) of intracellular free Ca2+ concentration ([Ca2+]i) induced by sperm in human oocytes (Ca2+ oscillations) were analyzed by confocal laser scanning microscopy and compared to Ca2+ oscillations induced in oocytes by the thiol reagent thimerosal. During the steady‐state period of sperm‐induced Ca2+ oscillations, each individual [Ca2+]i spike invariably began from a focus in oocyte periphery and spread throughout the entire peripheral region before propagating to the central ooplasm. This peripheral Ca2+ wave was immediately followed by an explosive [Ca2+]i increase in the central ooplasm. However, this central [Ca2+]i rise only peaked when [Ca2+]i in the peripheral ooplasm was already on the decline. Moreover, the peak [Ca2+]i values were always considerably higher in the oocyte center than in the periphery. In contrast, thimerosal‐induced Ca2+ oscillations did not show this particular form of propagation. These data show that sperm‐induced Ca2+ oscillations have a unique pattern of spatial dynamics and suggest that the bulk of Ca2+ mobilized during each spike is released from stores that have a relatively high threshold for Ca2+‐induced Ca2+ release (CICR). These stores are poorly developed, if not absent, in the oocyte cortex, and CICR from them is triggered by previous CICR from another type of store with a lower threshold that are preferentially located in the oocyte cortex and act as a detonator. © 1995 Wiley‐Liss, Inc.
Sry‐negative XX sex reversal in the American cocker spaniel dog
Authors: Meyers‐Wallen V. N. , Palmer V. L. , Acland G. M. , Hershfield B.
Published: February 2005

The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY‐negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y‐specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley‐Liss, Inc.
Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata)
Authors: Hyllner Sven Johan , Barber Hipolito Fernández‐Palacios , Larsson D. G. Joakim , Haux Carl
Published: February 2005

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol‐17β. As males of both species synthesize these proteins after treatment with estradiol‐17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley‐Liss, Inc.
Distribution of the intermediate filament proteins vimentin, keratin, and desmin in the bovine ovary
Authors: van den Hurk R. , Dijkstra G. , van Mil F. N. , Hulshof S. C. J. , van den Ingh Th. S. G. A. M.
Published: February 2005

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co‐localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore‐active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin‐positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley‐Liss, Inc.
Isolation, culture, and characterization of equine oviduct epithelial cells in vitro
Authors: Thomas Philip G. A. , Ignotz George G. , Ball Barry A. , Miller Paula G. , Brinsko Steven P. , Currie Bruce
Published: February 2005

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol‐17β or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers. © 1995 Wiley‐Liss, Inc.
Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization
Authors: Mozingo Nancy M. , Vacquier Victor D. , Chandler Douglas E.
Published: February 2005

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley‐Liss, Inc.
UV irradiation of chromosomal DNA and its effect upon MPF and meiosis in mammalian oocytes
Authors: Bradshaw J. , Jung T. , Fulka J. , Moor R. M.
Published: February 2005

Bovine oocytes were irradiated at germinal vesicle (GV) or metaphase II (MI) stage, after Hoechst staining, with chromosomally focused UV‐C (254 nm) or UV‐A (> 330 nm). UV‐C irradiation at GV stage did not inhibit germinal vesicle breakdown (GVBD) or chromosomal condensation; spindle formation was abolished and maturation promoting factor (MPF) levels failed to increase. UV‐A irradiation at GV stage caused meiotic arrest at anaphase I; MPF levels were lower than control. UV‐C irradiation at MII stage led to subsequent abnormal parthenogenetic activation when MPF levels failed to decrease. A normal male but no female pronucleus was formed at fertilization. UV‐A irradiation at MII stage also caused abnormal activation; MPF levels declined normally. A normal male and abnormal female pronucleus formed at fertilization. UV‐A irradiation results have implications for oocyte evaluation during development using Hoechst staining. UV‐C irradiation is a potential means for oocyte enucleation in nuclear transfer. © 1995 Wiley‐Liss, Inc.
F‐actin is involved in control of bovine cumulus expansion
Authors: Šutovský Peter , Fléchon Jacques E. , Pavlok Antonín
Published: February 2005

Previously, we showed that the gonadotropin‐induced expansion of bovine cumulus oophorus occurs concomitantly with the rearrangement of micro‐filaments (MFs) inside cumulus cell cytoplasm (Šutovský et al., 1993: Biol Reprod 49:1277–1287; Šutovský et al., 1994: Reprod Nutr Dev 34:415–425) and that cumulus expansion in cattle is accompanied by the increased expression of extracellular matrix (ECM) glycoproteins laminin and type IV collagen as well as of their actin‐linked membrane receptors, integrin subunits α‐6 and β‐1 (Šutovský and Motlik: 1994). The present study was undertaken to determine the spatial and temporal relationship between cytoskeletal rearrangement and ECM synthesis during cumulus expansion. Using electron microscopy and confocal (LSCM) and conventional fluorescence microscopy, we compared the expression of the above integrins and ECM proteins and the rearrangement of cytoskeleton in the gonadotropin‐stimulated bovine oocyte cumulus complexes (OCCs) with those exposed to gonadotropin stimulation and to ECM synthesis inhibitor 6‐diazo‐5‐oxo‐L‐norleucin (DON), or MF‐disorganizing drug cytochalasin B (CB). In control OCCs, the 24‐hr culture in the presence of follicle stimulating hormone/luteinizing hormone (FSH/LH) caused the expansion of cumuli oophori and an extensive rearrangement of MFs in the cytoplasm of cumulus cells. Concomitantly, we observed an increased deposition of laminin and type IV collagen in the intercellular spaces among cumulus cells. The redistribution of microtubules (MTs), intermediate filaments (IFs), and integrin chains α‐6 and p‐1 also occurred at this time. The addition of 20 μg/ml of CB prevented cumulus expansion and accumulation of laminin and type IV collagen in the OCCs. Moreover, cytochalasin treatment blocked the redistribution of MTs and IFs, and caused the disorganization of MFs and dispersion of integrins in cumulus cells. In contrast, the distribution of integrins and cytoskeletal elements was not affected when we blocked cumulus expansion and ECM protein accumulation by DON. These data suggest that F‐actin acts upstream of ECM synthesis in the cascade of events leading to the expansion of bovine cumulus ooophorus. © 1995 Wiley‐Liss, Inc.
Molecular cloning, tissue distribution, and hormonal control in the ovary of Cx41 mRNA, a novel Xenopus connexin gene transcript
Authors: Yoshizaki Goro , Patiño Reynaldo
Published: February 2005

Gap junctions (GJ) are aggregates of intercellular channels, composed of connexin (Cx) protein, between adjacent cells. The vertebrate ovarian follicle contains homocellular (granulosa cell‐granulosa cell) and heterocellular (granulosa cell‐oocyte) GJ. However, the function of GJ during final oocyte differentiation (maturation) is controversial. The objectives of this study are to reexamine the number and identity of Cx genes that are expressed in the Xenopus ovary, and to examine the potential role of GJ in oocyte maturation by determining the temporal association between changes in ovarian Cx mRNA content and the process of maturation. We used reverse transcriptase‐polymerase chain reaction to amplify ovarian cDNA fragments using degenerate Cx primers. We amplified three Cx‐like fragments: one was novel and two corresponded to known Cx of Xenopus ovaries (Cx38 and 43). The novel fragment was used to screen an ovarian cDNA library. One positive clone was identified and its nucleotide sequence determined. Its deduced amino acid sequence showed that it corresponded to a novel Cx, Cx41, belonging to the Group II class of Cx. Xenopus Cx41 showed the highest homology to rat Cx37 (65% identity, 80% similarity). Also, the last 10 C‐terminal amino acids of Cx41 were identical to those of rat, mouse, and human Cx37. Cx41 transcripts were detected by riboprobe mapping in ovarian somatic cells, heart, leg muscle, liver and eye, but not in brain or in oocytes of any developmental stage. Full‐grown follicles incubated in vitro with human chorionic gonadotropin became committed to mature within 1–4 hr, and physical signs of maturation (germinal vesicle breakdown) were seen at 4–5 hr. Significant reductions in the levels of Cx41 and 43, but not 38 transcripts were seen at 4 hr, after oocytes had committed to mature. Thus, if availability of Cx mRNA determines availability of Cx protein and GJ, our results would suggest that irreversible commitment to maturation occurred prior to major declines in follicular GJ during the periovulatory period. The present study is the first to report the presence of at least two hormone‐responsive Cx gene transcripts (Cx41 and 43 in Xenopus) in ovaries of a single animal species. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.