bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+‐Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis
Authors: Fraser Lynn R. , Abeydeera L. R. , Niwa K.
Published: February 2005

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca2+ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: “F,” characteristic of uncapacitated, acrosome‐intact cells; “B,” characteristic of capacitated, acrosome‐intact, cells; “AR,” characteristic of capacitated, acrosome‐reacted cells. Over a 60‐min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently ∼ 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells. In the presence of either quercetin, a Ca‐ATPase inhibitor used at 50–200 μM, or W‐7, a calmodulin antagonist used at 5–125 μM, capacitation per se was accelerated, as evidenced by significant decreases in F and significant increases in B pattern cells; only the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 μM W‐7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na+ ionophore monensin at 500 μM significantly accelerated both capacitation and acrosomal exocytosis. The addition of the dihydropyridine calcium channel blocker nifedipine at 10 nM, just prior to monensin, did not inhibit capacitation (F to B transition) but blocked acrosomal exocytosis (B to AR transition). We suggest that Ca2+ is required for functional changes in bull sperm, with a Ca2+‐ATPase modulating intracellular Ca2+ during capacitation and calcium channels controlling the Ca2+ influx required for acrosomal exocytosis. © 1995 Wiley‐Liss, Inc.
Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA‐Acr.1 with similarities to the SP‐10 antigen
Authors: Beaton Sandra , Have José ten , Cleary Andrew , Bradley Mark P.
Published: February 2005

We have isolated and characterized a cDNA, cFSA‐Acr.1, encoding a testis‐specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA‐Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP‐10, and the mouse sperm protein, MSA‐63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP‐10, and mouse MSA‐63 sequences. In addition to cFSA‐Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA‐Acr proteins. © 1995 Wiley‐Liss, Inc.
Inactivation of histone H1 kinase by Ca2+ in rabbit oocytes
Authors: Collas Philippe , Chang Thomas , Long Charles , Robl James M.
Published: February 2005

The present study investigated the role of intracellular Ca2+ (Ca2+i) elevation on the inactivation of maturation promoting factor (MPF) in rabbit oocytes. The effects of the number of Ca2+ stimulations and of the amplitude of Ca2+i elevation on the profile of histone H1 kinase activity were determined. A Ca2+ stimulation consisted of transferring mature oocytes from culture medium to 0.3 M mannitol containing 0.1–1.0 mM CaCl2, and pulsing them at 1.25 kV/cm for 10 μsec, or microinjecting 2–8 mM CaCl2 into the oocyte cytoplasm. The number of electrically‐induced Ca2+ stimulations was varied, and amplitude of the Ca2+i rise was controlled by altering Ca2+ concentration in the pulsing medium or the injection pipette. Ca2+i concentration was determined with fura‐2 dextran; oocytes were snap‐frozen at indicated time points and assayed for H1 kinase activity. The activity was quantified by densitometry and expressed as a fraction of activity in nonstimulated oocytes. Electrically‐mediated Ca2+i rises inactivated H1 kinase in a manner dependent on the number of Ca2+ stimulations. A single Ca2+ stimulation inactivated H1 kinase to 30–40% of its initial activity. However, H1 kinase inactivation was only transient, regardless of the amplitude of the electrically‐ or injection‐mediated Ca2+i elevation. Increasing the number of Ca2+ stimulations helped to maintain H1 kinase activity at basal (pronuclear) levels. The results show the necessity of a threshold of Ca2+i concentration to trigger MPF inactivation, and suggest a role for the extended period of time over which Ca2+i oscillates at fertilization. © 1995 Wiley‐Liss, Inc.
Production of live offspring with predicted genotypes using PCR‐RFLP analysis of polar bodies from mouse oocytes
Authors: Wheeler M. B. , Noble J. A. , Jarrell V. L.
Published: February 2005

Accurate identification of genotypes in gametes and early embryos could facilitate the efficient production of offspring with desirable traits. This study demonstrates the feasibility of producing offspring with predictable genotypes from micromanipulated mouse oocytes. The Polymerase Chain Reaction (PCR) was used to amplify genes in the IA subregion of the major histocompatibility complex of the mouse. The validity of the approach was demonstrated in experiment 1 with IA haplo‐types of unfertilized mouse ova amplified via PCR and distinguished by restriction fragment length polymorphism (RFLP) analysis. In experiment 2, fertilized oocytes were micromanipulated to remove the first and second polar bodies, which were then genotyped by validated PCR‐RFLP procedures. Primary oocytes of heterozygous females contain two copies of each of the different alleles. Following meiosis I and II, the genotype of the ovum was predicted by subtracting the alleles observed in micromanipulated polar body samples. Sixty‐two fertilized ova were micromanipulated and transferred to recipient females resulting in 27 live offspring (44%). The correct maternal contribution to the embryonic genotype was predicted in 19 of 27 (71%) offspring as confirmed by PCR‐RFLP analysis of DNA from pup tails. Predicted genotypes of two pups were not confirmed (7%), whereas no prediction could be made in six cases (22%). © 1995 Wiley‐Liss, Inc.
Initial culture behaviour of rat blastocysts on selected feeder cell lines
Authors: Ouhibi Nadia , Sullivan Neil F. , English Jennifer , Colledge William H. , Evans Martin J. , Clarke Neil J.
Published: February 2005

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst‐derived cell lines, we examinated the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3–6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo‐derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial‐like cells were isolated on REF and colonies of ES‐like cells on the RUCs. The ES‐like cells were positive for expression of alkaline phosphatase and stage‐specific embryonic‐antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat. © 1995 Wiley‐Liss, Inc.
DNA Synthesis and pronucleus development in pig zygotes obtained in vivo: An autoradiographic and ultrastructural study
Authors: Laurnick J. , Hyttel P. , Kopecny V.
Published: February 2005

Porcine zygotes flushed from oviducts 48,52,56,60, or 64 hr after hCG were incubated 30 min in 3H‐thymidine, transferred to nonradioactive medium for 2 hr, and incubated for 30 min with 14C‐thymidine. After this procedure, ova were prepared (i.e., at 51,55,59,63, or 67 hr after hCG) for autoradiography and ultrastructural observations, respectively.

The first autoradiographic labelling, i.e., DNA synthesis, was observed at 56–56.5 hr after hCG, while the latest labelling was seen at 60–60.5 hr.

At 51 hr after hCG, formation of the pronuclear envelope was observed, while no nucieolus precursor bodies or prestages to these structures were found. At 55 hr a few clusters of small electron‐dense granules were observed, together with condensed chromatin in the pronuclei. At 59 hr the apposed regions of both pronuclei contained nucleolus precursor bodies and condensed chromatin, in close contact with both clusters of small granules and clusters of an additional category of large granules and the nuclear envelope. Additionally, large accumulations of the small granules were found in the vicinity of similarly sized accumulations of the large granules without chromatin association. At 63 hr the spherical accumulations large granules on some occasions presented a central vacuole, and condensed chromatin and clusters of small granules were attached to its periphery. Within the vacuole, electrondense material was found.

It is concluded that (1) the S‐phase in porcine zygotes is initiated around 56 hr post‐hCG injection and is of a duration of 4.5–7.5 hr and (2) the progress of the S‐phase is paralleled by the appearance of and complex interaction between different granules in the nucleoplasm. © 1995 Wiley‐Liss, Inc.
PRINS as a method for rapid chromosomal labeling on human spermatozoa
Authors: Pellestor Franck , Girardet Anne , Lefort Geneviève , Andréo Brigitte , Charlieu Jean Paul
Published: February 2005

Direct in situ labeling of human spermatozoa was performed using the PRINS method. This technique is based on annealing of specific oligonucleotide primers, and subsequent primer extension by a Taq DNA polymerase. The reaction was carried out on a programmable temperature cycler, and labeling was obtained in a 1‐hr reaction. The method was successfully tested with specific primers for chromosomes 13, 16, and 21. This suggests that PRINS may be a fast and reliable technique for detecting aneuploidies. © 1995 Wiley‐Liss Inc.
Identification of heterotrimeric G proteins in human sperm tail membranes
Authors: Hinsch Klaus‐Dieter , Schwerdel Carola , Habermann Barbara , Schill Wolf‐Bernhard , Müller‐Schlösser F. , Hinsch Elvira
Published: February 2005

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α‐subunits, and A 86, which detects all known pertussis toxin‐sensitive α‐subunits, reacted specifically with a 40‐kDa protein. Antisera against individual G protein α‐subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2‐subunit of G proteins, identified a 35‐kDa protein in sperm tail membranes. Antisera against the 36‐kDa β1‐subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α‐subunits nor G protein β‐subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin‐sensitive α‐subunits. However, membrane preparations of nonpurified human spermatozoa contained α2 subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α‐subunits; the putative β‐subunit was identified as a β2‐subunit. © 1995 Wiley‐Liss, Inc.
Induction of paternal genome loss by the paternal‐sex‐ratio chromosome and cytoplasmic incompatibility bacteria (Wolbachia): A comparative study of early embryonic events
Authors: Reed Kent M. , Werren John H.
Published: February 2005

Paternal genome loss (PGL) during early embryogenesis is caused by two different genetic elements in the parasitoid wasp, Nasonia vitripennis. Paternal sex ratio (PSR) is a paternally inherited supernumerary chromosome that disrupts condensation of the paternal chromosomes by the first mitotic division of fertilized eggs. Bacteria belonging to the genus Wolbachia are present in Nasonia eggs and also disrupt paternal chromosome condensation in crosses between cytoplasmically incompatible strains. Cytoplasmic incompatibility Wolbachia are widespread in insects, whereas PSR is specific to this wasp. PGL results in production of male progeny in Nasonia due to haplodiploid sex determination. The cytological events associated with PGL induced by the PSR chromosome and by Wolbachia were compared by fluorescent light microscopy using the fluorochrome Hoescht 33258. Cytological examination of eggs fertilized with PSR‐bearing sperm revealed that a dense paternal chromatin mass forms prior to the first metaphase. Quantification of chromatin by epifluorescence indicates that this mass does undergo replication along with the maternal chromatin prior to the first mitotic division but does not replicate during later mitotic cycles. Contrary to previous reports using other staining methods, the paternal chromatin mass remains condensed during interphase and persists over subsequent mitotic cycles, at least until formation of the syncytial blastoderm and cellularization, at which time it remains near the center of the egg with the yolk nuclei. Wolbachia‐induced PGL shows several marked differences. Most notable is that the paternal chromatin mass is more diffuse and tends to be fragmented during the first mitotic division, with portions becoming associated with the daughter nuclei. Nuclei containing portions of the paternal chromatin mass appear to be delayed in subsequent mitotic divisions relative to nuclei free of paternal chromatin. Crosses combining incompatibility with PSR were cytologically similar to Wolbachia‐induced PGL, although shearing of the paternal chromatin mass was reduced. Wolbachia may, therefore, block an earlier stage of paternal chromatin processing in the fertilized eggs than does PSR. © 1995 Wiley‐Liss, Inc.
Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods
Authors: Dinnyés A. , Wallace G. A. , Rall W. F.
Published: February 2005

We examined possible genotype effects on the survival of 8‐ to 16‐cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P >0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18–19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR♀ X B6D2F1 ♂ embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post‐implantation developmental potential and not their sensitivity to cryoinjury. © 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.
IPEB transcription factor regulating the intracisternal a particle gene during F9 cell differentiation is expressed at sites of lymphoid development
Authors: Kamat Jaya P. , Basu Amitabha , Satyamoorthy Kapaettu , Xu Mei Q. , Showe Louise , Howe Chin C.
Published: February 2005

The murine intracisternal A particle (IAP) proviral elements are expressed at low levels in undifferentiated F9 embryonal carcinoma cells but are highly expressed when F9 cells are induced to differentiate into parietal endoderm‐like cells. IAP elements are also expressed in parietal endoderm‐like PYS‐2 cells. We previously identified an IAP proximal enhancer (IPE) element that mediates a F9 differentiation‐specific enhancer activity. We also identified a 60 kDa IPE binding (IPEB) protein whose activity is high in PYS‐2 cells, where IAP is expressed, but very low in F9 cells.

Transcription of IAP elements has also been shown in the adult mouse thymus and in activated splenic B cells. We have now shown by DNA affinity chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and band‐shift analysis that the 60 kDa IPEB is expressed in adult T lymphocytes and in resting as well as lipopolysaccharide activated splenic B cells but not in adult liver cells, suggesting an important role for IPEB in IAP transcription in vivo. In addition, we find IPEB expressed in the fetal mouse at sites of lymphoid development, such as the liver, spleen, and thymus, suggesting it may play an important role in gene expression during lymphoid development. In support of this, we find IPEB in the human T cell tumor lines, Jurkat and Molt 13, as well as the Daudi B cell line and in the normal calf thymus and in the thymus and spleen of the chicken and rat. © 1995 Wiley‐Liss, Inc.
Strain‐specific progression of α‐amanitin—treated mouse embryos beyond the two‐cell stage
Authors: Rambhatla Lakshmi , Latham Keith E.
Published: February 2005

Mouse embryos produced by the fertilization of eggs from (B6D2)F1 and CF‐1 mice differ in their ability to complete the second cell cycle in the presence of α‐amanitin. Essentially all embryos obtained from CF‐1 mothers arrest at the two‐cell stage when cultured from the late one‐cell stage in α‐amanitin at concentrations that prevent zygotic genome activation, while up to 15% of the embryos obtained from (B6D2)F1 mothers can progress to the three‐ to four‐cell stage. This occurs even at α‐amanitin concentrations that are fivefold greater than that required to prevent gene transcription. We propose that eggs of certain strains of mice may be endowed with greater supplies of macromolecules to support early development and that a percentage of these embryos can complete the second cell cycle in the absence of transcription. This difference may contribute to the strain‐dependent differences in development in vitro. © 1995 Wiley‐Liss, Inc.
Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes
Authors: Blondin Patrick , Sirard Marc‐André
Published: February 2005

Follicular size, follicular atresia, and oocyte morphology were investigated for the possible relation of these characteristics to the developmental competence of bovine oocytes. Ovaries from a local slaughterhouse were dissected to obtain a heterogeneous population of follicles. Half of each follicle was fixed for histological analysis, and the oocytes were detached carefully and cultured individually. Before in vitro maturation, the oocytes were grouped into six different classes based on the morphology of the cumulus and the ooplasm: classes 1 and 2 represent oocytes with a homogeneous ooplasm plus a compact and complete cumulus, and classes 3–6 represent oocytes with a granulated ooplasm and an incomplete and/or expanded cumulus. Oocytes from class 3 (beginning of expansion in outer cumulus layers and slight granulations in the ooplasm) developed past the 16‐cell stage significantly (P<0.05) more than oocytes with a compact and complete cumulus (classes 1 and 2) and oocytes from classes 4–6 (incomplete and/or expanded cumulus) after 5 days of in vitro culture. Oocytes from follicles measuring 3 mm or less did not develop past the 16‐cell stage, whereas follicles of 3–5 mm and 5 mm or larger developed at similar rates (17% and 21% morulae, respectively). The state of the follicle did not affect whether an embryo reached at least the 16‐cell stage, as comparable rates were obtained in all three groups of follicles: nonatretic (20%), intermediate (14%), and slightly atretic (16%). We concluded that oocytes acquire developmental competence late in the follicular phase, possibly when the first signs of atresia have appeared, and that oocytes with beginning signs of degeneration (class 3) will develop significantly more than all other classes. Class 3 oocytes originated from follicles that were generally atretic and therefore in later phases of follicular growth, suggesting that these oocytes, having been subjected longer to the follicular microenvironment, are more differentiated (possibly at the cytoplasmic level) than other classes of oocytes. © 1995 Wiley‐Liss, Inc.
Ultrastructural localization of acrosome reaction‐inducing substance (ARIS) on sperm of the starfish Asterias amurensis
Authors: Longo Frank J. , Ushiyama Akira , Chiba Kazuyoshi , Hoshi Motonori
Published: February 2005

Using colloidal gold tagged ligands we have identified the ultrastructural site of ARIS binding to intact and acrosome‐reacted starfish sperm. In intact sperm, colloidal gold conjugated ARIS was specifically localized to a single domain (0.1–0.3μ in diameter) on the plasma membrane. This site was located on the anterior‐lateral aspect of the sperm head, that is, just peripheral to the region occupied by the acrosomal vesicle and periacrosomal components. When sperm were labeled with colloidal gold conjugated ARIS, washed to remove unbound label, and then induced to undergo the acrosome reaction, the labeled patch remained associated with the plasma membrane and was positioned just lateral to the acrosomal process. However, when sperm were suspended in labeled ARIS and induced to undergo the acrosome reaction, label was observed along the entire anterior aspect of the sperm head with the exception of the acrosomal process. Labeling along the entire anterior aspect of the sperm head in this case was deemed to be nonspecific and due to binding of colloidal gold tagged molecules to components formerly located within the acrosomal vesicle, as the same pattern was obtained using colloidal gold tagged bovine serum albumin. Quantitative and qualitative aspects of ARIS binding observed here by electron microscopy are in agreement with measured binding characteristics previously reported (Ushiyama et al., 1993a: Zygote 1:121–127; Ushiyama et al., 1993b: J Reprod Dev 39:53–54), and indicate that the site of labeled ARIS binding represents a specific plasma membrane domain occupied by ARIS receptors. © 1995 Wiley‐Liss, Inc.
Cloning of a novel rat placental prolactin‐like protein C‐related cDNA
Authors: Conliffe Phyllis R. , Simmen Rosalia C. M. , Buhi William C. , Shiverick Kathleen T.
Published: February 2005

Prolactin‐like protein C (PLP‐C) is a major rat placental protein which is expressed during the second half of pregnancy and belongs to the growth hormone‐prolactin family. Here we report on the isolation of overlapping rat placental cDNAs which specify a transcript of 915 base pairs and predict a 205‐amino acid translated product. The full‐length cDNA shares 93% homology with the nucleotide sequence reported for PLP‐C, and the putative protein, which we designate PCRP (prolactin‐like protein C‐related protein), exhibits 88% homology with the PLP‐C precursor protein. PCRP lacks the signal sequence and the first 2 N‐terminal cysteine residues present in PLP‐C. Northern blot analysis indicated the basal zone‐specific expression of PCRP mRNA, with no detectable expression in decidua and labyrinth. Southern blot analysis of rat genomic DNA using PCRP cDNA as a probe demonstrated multiple hybridization bands, suggestive of a family of genes encoding prolactin‐like proteins. Western immunoblot analysis of basal zone culture media using a PCRP antipeptide antiserum revealed at least 5 immunoreactive proteins. The existence of a PLP‐C family of proteins in rat placenta after midpregnancy suggests their functional significance in the maintenance of pregnancy and fetal development. © 1995 Wiley‐Liss, Inc.
Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti‐P26h hamster sperm protein
Authors: Bégin Sylvie , Bérubé Bruno , Boué Franck , Sullivan Robert
Published: February 2005

The binding of the spermatozoon to the zona pellucida is a species‐specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26‐kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species‐specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti‐P26h antibodies in a sperm‐zona pellucida assay using hamster and mouse gametes. Anti‐P26h IgGs inhibit, in a dose‐dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti‐26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm‐zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti‐P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley‐Liss, Inc.
Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly
Authors: Moses Ruth M. , Kline Douglas
Published: February 2005

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization‐induced increase in intracellular calcium concentration ([Ca2+]i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca2+]i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca2+]i, and despite disruption of the Spindle. © 1995 Wiley‐Liss, Inc.
Detection and location of amphiregulin and Cripto‐1 expression in the developing postnatal mouse mammary gland
Authors: Kenney N. J. , Huang R.‐P. , Johnson G. R. , Wu J.‐X. , Okamura D. , Matheny W. , Kordon E. , Gullick W. J. , Plowman G. , Smith G. H. , Salomon D. S. , Adamson E. D.
Published: February 2005

Amphiregulin (Ar) and Cripto‐1 (Cr‐1) are growth promoting peptides that share amino acid sequence homology with epidermal growth factor (EGF). The present study examined Ar and Cr‐1 mRNA and protein expression during various stages of C57BL/6 mouse mammary morphogenesis. Reverse transciption‐polymerase chain reaction (RT‐PCR) was used to detect transcripts for Ar and Cr‐1 at all stages of mammary development. Immunocytochemical (ICC) localization demonstrated that in virgin 4‐week to mature 12‐week‐old mouse fourth inguinal mammary gland, Ar and Cr‐1 are expressed in the stromal cells, luminal epithelial cells, and myoepithelial cells of the branching ducts. Ar, and to lesser extent Cr‐1, were also found in the epithelial cap cells and in the luminal epithelial cells of the advancing terminal end bud (TEB) from virgin 4‐week and 6‐week‐old mice. Western blot analysis demonstrated that both Ar (28 and 26 kDa) and Cr‐1 (90, 67, 56, and 21 kDa) proteins are expressed in virgin, 13.5 day midpregnant and in the 14 day lactating mammary gland. In addition, Ar and Cr‐1 are associated with developing alveolar structures as determined by ICC. These results imply that together with EGF and transforming growth factor alpha (TGFα), Ar and Cr‐1 may play salient roles as modifiers in the morphogenesis and differentiation of the mammary gland. © 1995 Wiley‐Liss, Inc.
Analysis of G protein α subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT‐PCR approach
Authors: Rambhatla Lakshmi , Patel Bela , Dhanasekaran N. , Latham Keith E.
Published: February 2005

We have developed a novel reverse transcription‐polymerase chain reaction (RT‐PCR)‐based approach for systematically quantifying in a single experiment the abundances of many different mRNAs in preimplantation mouse embryos. With this approach, the entire mRNA population from a small number of embryos is amplified while preserving the relative abundance of each mRNA in the cDNA population. The cDNA is analyzed by quantitative hybridization to radiolabeled probes. The approach is very sensitive and provides reliable, quantitative data regarding changes in mRNA abundance. A major advantage of this method is that estimates of mRNA copy number can be obtained and compared between different mRNAs. With this approach, we analyzed the patterns of expression of nine G protein α subunit mRNAs (Gαs, Gαi, Gαq′, Gαo′, and Gα11–15) in oocytes, eggs, and preimplantation embryos from fertilization to the blastocyst stage. Six α subunit mRNAs were expressed at significant levels, all of which underwent significant temporal alterations in expression. The mRNAs encoding some α subunit types were expressed predominantly in the egg and 1‐cell embryo, underwent sharp reductions during the 2‐cell stage, and were re‐expressed between the 8‐cell and blastocyst stages. One α subunit mRNA increased in abundance at the early blastocyst stage. The possible significance of these alterations in G protein mRNA abundance to embryonic development is discussed. © 1995 Wiley‐Liss, Inc.
Separation of round spermatids from the rat using an immunoselection panning technique
Authors: Pelengaris S. A. , Moore H. D. M.
Published: February 2005

A method was devised for the isolation of round spermatids from the rat using a positive immunoselection technique (panning). A testis suspension was prepared from adult rats by enzymatic digestion of seminiferous tubules with collagenase. Specific mouse monoclonal antibody (97.25) was indirectly attached to Petri dishes and used in a panning protocol to purify spermatids from the testis cell suspension. The quantity and purity of cells isolated were determined by cell counts and histochemical (periodic acid‐Schiff stain) or by immunostaining with acrosome‐specific antibodies. A mean yield of 1.38 ± 0.15 × 107 cells per dish was obtained with a purity of more than 90%. The viability of the cells was confirmed by epifluorescent microscopy with propidium iodide/carboxyfluorescein acetate probes. Northern blot analysis of RNA extracted directly from the dish indicated good integrity of a spermatid‐specific transcript of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). © 1995 Wiley‐Liss, Inc.
Composition of human zona pellucida as revealed by SDS‐PAGE after silver staining
Authors: Bercegeay S. , Jean M. , Lucas H. , Barriere P.
Published: February 2005

The macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by one‐ and two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1‐D‐ and 2‐D‐SDS‐PAGE) under reducing conditions after silver staining. ZP specimens obtained after in vitro fertilization were removed from pools of heavily fragmented embryos and inseminated oocytes that failed to fertilize. For unfertilized oocytes, two major bands with an apparent molecular weight of, respectively, 96 and 76–54 kDa were observed after 1‐D‐SDS‐PAGE and silver staining. When ZP were isolated from fragmented embryos, the electrophoretic pattern showed a marked attenuation of the 96‐kDa band. Silver‐stained 2‐D‐SDS‐PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 (Mr = 92–80 kDa, pl = 4.9–5.9), ZP2 (Mr = 66–58 kDa, pl = 5.0–6.0), ZP3H (high) (Mr = 72–58 kDa, pl = 3.5–5.1), and ZP3L (low) (Mr = 62–54 kDa, pl = 3.5–5.1). The human ZP3 family (ZP3H and ZP3L) showed marked heterogeneity. Fertilization‐associated changes were apparent in the electrophoretic pattern. ZP1 (Mr = 92–86 kDa, pl = 5.0–5.8) displayed a dramatic decrease in intensity, and a new component had migrated to a position similar to that of ZP2. This modification may have been responsible for one aspect of the zona reaction, and could have contributed to a zona block to polysperma. © 1995 Wiley‐Liss, Inc.
Effect of coculturing spermatozoa with oviductal cells on the incidence of polyspermy in pig in vitro fertilization
Authors: Dubuc A. , Sirard M.‐A.
Published: February 2005

It is known that oviductal cells play an important role in fertilization in vivo. We were interested in the effect of those cells on spermatozoa and their influence on the incidence of polyspermy in pig in vitro fertilization (IVF) when cocultured with spermatozoa. Oviductal cells are believed to select a highly fecund population of spermatozoa. By coculturing spermatozoa with oviductal cells it is possible to reduce the number of spermatozoa confronting the eggs at fertilization, reducing the incidence of polyspermy. In our study, spermatozoa were cocultured with oviductal cells for 30 min so that they could bind to the oviductal cells. Both bound and unbound spermatozoa were used for fertilization. The results show that the spermatozoa that were bound to the oviductal cells were capable of fertilizing the eggs, but the nonbound spermatozoa had a reduced penetration incidence. With the bound sperm, polyspermy incidence decreased and the two‐pronuclei proportion increased. We also found that with time the spermatozoa released from the cells had better motility than those that did not bind. Therefore, it is our belief that oviductal cells can be used for porcine IVF resulting in a lower polyspermy incidence and higher pronuclei incidence. © 1995 Wiley‐Liss, Inc.
Developmental control of transcription of the cat reporter gene by a truncated mouse alphafetoprotein gene regulatory region in transgenic mice
Authors: Ghebranious N. , Knoll B. J. , Yavorkovsky L. , Ilic Z. , Papaconstantinou J. , Lozano G. , Sell S.
Published: February 2005

A truncated mouse alphafetoprotein (AFP) gene promoter/enhancer region was tested for its ability to regulate the expression of theEscherichia colichloramphenicol acetyltransferase (CAT) reporter gene in the livers of transgenic mice. The AFP regulatory region lacked any AFP gene structural DNA, included one enhancer sequence together with the proximal promoter sequence, and an element believed to be responsible for the postnatal repression of AFP gene transcription. The neonatal livers of AFP/CAT transgenic mice showed a high level of CAT enzyme expression, which was dramatically reduced between 7 and 14 days after birth. The staining of liver sections with anti‐CAT antibodies showed that this expression was limited to hepatocytes. In one lineage, reexpression of CAT in the adult liver could be achieved by restitutive proliferation of hepatocytes following partial hepatectomy or CCl4‐induced necrosis; reexpression in young animals (3–4 weeks of age) was even greater. These studies show that a truncated AFP promoter/enhancer region functions in a tissue‐specific and developmental stage‐specific fashion, and may be used to control the expression of other genes in the livers of transgenic mice. © 1995 Wiley‐Liss, Inc.
In vitro pluripotency of epiblasts derived from bovine blastocysts
Authors: Talbot Neil C. , Powell Anne M. , Rexroad Caird E.
Published: February 2005

Two experiments were conducted to compare the utility of in vitro‐ and in vivo‐derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo‐collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro‐produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo‐collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro‐produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte‐like cells, and macrophage‐like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo‐derived blastocysts, especially from early‐hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley‐Liss, Inc.

This artilce is a US Government work and, as such, is in the public domain in the United States of America.
Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning
Authors: Zakhartchenko V. , Wolf E. , Palma G. A. , Brem G.
Published: February 2005

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25–34), intermediate (40–55), and high (60–81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50–83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40–55) or high (60–81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40–55 cells) and 41% versus 25% (60–81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning. © 1995 wiley‐Liss, Inc.
Cloning of complementary DNA encoding a 135‐kilodalton protein secreted from porcine corpus epididymis and its identification as an epididymis‐specific α‐mannosidase
Authors: Okamura Naomichi , Tamba Michiko , Liao Hong‐Jun , Onoe Sinji , Sugita Yoshiki , Dacheux Françloise , Dacheux Jean‐Louis
Published: February 2005

In the preceding study (Okamura et al., 1992; Biol Reprod 47:1040–1052) we suggested that a 135‐kDa protein secreted by porcine epididymis is involved in the sperm maturation. In this work, we have isolated the cDNA clone coding the 135‐kDa protein in an effort to investigate its structure and function.

The 135‐kDa protein was purified from porcine cauda epididymal fluid. Three oligonucleotide probes were synthesized according to the amino acid sequences of N‐termini of the native protein and trypsin‐digested peptides. A cDNA clone hybridizing with these three probes was isolated from the cDNA library derived from the porcine proximal corpus epididymis. It encodes a novel protein with 1,006 amino acid residues in an open reading frame.

Its overall amino acid sequence was significantly homologous (25.7%) to the α‐mannosidase precursor of Dictiostelium discoideum (P34098). The 135‐kDa protein could digest both p‐nitro‐phenyl‐α‐D‐mannoside and high mannose oligo saccharide (Man8‐GlcNAc2), strongly suggesting that it is an α‐mannosidase homologue.

The expression of this protein was specific to porcine and was localized to the very narrow parts of epididymis: the border of the caput and corpus epididymis. This protein may serve as a good marker for the functional differentiation in porcine epididymis. A possible role of this protein in the species‐specific sperm‐egg interaction is discussed. © 1995 wiley‐Liss, Inc.
Analysis of stimulatory and inhibitory amino acids for development of hamster one‐cell embryos in vitro
Authors: McKiernan Susan H. , Clayton Murray K. , Bavister Barry D.
Published: February 2005

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1‐cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium‐5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM‐6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM‐5 and −6 (chemically defined, protein‐free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley‐Liss, Inc.
Messenger RNA encoding an estrogen‐dependent oviduct secretory protein in the sheep is localized in the apical tips and basal compartments of fimbria and ampulla epithelial cells implying translation at unique cytoplasmic foci
Authors: Murray Mary K. , DeSouza Mary M.
Published: February 2005

Data from our laboratory have shown that an estrogen (E2)‐dependent Mr 90,000–92,000 protein and its mRNA are synthesized and expressed in abundant amounts at estrus from the fimbria and ampulla, not isthmus, oviduct of the sheep. Immunocytochemical studies have shown that the Mr 90,000–92,000 protein is contained in apical secretory granules of oviduct epithelial cells. The objective of this study was to determine whether the mRNA for the E2‐dependent oviduct protein was localized and compartmentalized in similar manner. Fimbria, ampulla, and isthmus oviducts obtained from estrous ewes were flash frozen in liquid nitrogen, cryosectioned, fixed in 4% paraformaldehyde, hybridized with digoxigenin (DIG)‐labeled oviduct‐specific riboprobes, incubated in anti‐DIG antibodies conjugated with alkaline phosphatase, and developed in color substrate. Oviduct protein‐specific transcripts were localized to basal perinuclear compartments and, surprisingly, at sites distant from the nucleus in the apical cytoplasm of epithelial cells in the fimbria and ampulla. No specific reaction product was observed in the underlying mucosa or smooth muscle layers. Oviduct protein mRNA was contained predominantly in the apical cytoplasm of epithelial cells at the free margins of mucosal folds and in the basal regions of cells located at the crypts of longitudinal folds. No reaction product was present when sections of the fimbria and ampulla oviduct of estrous ewes were incubated in sense riboprobe to the oviduct protein. In addition, when sections of the isthmus oviduct obtained from estrous ewes or fimbria and ampulla oviducts from long‐term ovariectomized ewes were hybridized with antisense riboprobes no specific reaction product was detected. Electron microscopy of oviduct protein mRNA containing areas revealed the presence of secretory granules, rough endoplasmic reticulum (RER) and Golgi in the apical cytoplasm, and RER in the basal regions of epithelial cells. These data show that the mRNA encoding an E2‐dependent oviduct‐specific protein is distributed in epithelial cells at perinuclear foci and at sites distant from the nucleus, which are also the sites of protein localization and protein synthesizing organelles, implying translation at unique cytoplasmic foci. © 1995 wiley‐Liss, Inc.