bluuebunny/crossref_metadata_2025 huggingface dataset:
The incidence of aneuploidy after single pulse electroactivation of mouse oocytes
Authors: Henery C. C. , Kaufman M. H.
Published: February 2005

A brief electric pulse often produces a high rate of activation of recently ovulated oocytes. Some other efficient parthenogenetic stimuli, such as alcohol, however, disrupt the spindle apparatus and increase the incidence of aneuploidy. In this paper, we have determined whether electroactivation per se increases the incidence of chromosomal segregation errors in haploid parthenogenones as evidenced at first cleavage mitosis.

Superovulated F1 hybrid female mice were killed at 15.5, 18.5, 22.5, and 25 h after the HCG injection. Batches of 10–12 cumulus‐denuded oocytes were transferred to an electroactivation chamber containing mannitol which was connected to a high voltage pulse stimulator and the pulse was triggered once. A high proportion of oocytes activated following this treatment, but only the single‐pronuclear haploid parthenogenones were incubated overnight in medium containing colcemid, to determine the incidence of aneuploidy as evidenced at first cleavage mitosis.

“Sham” electroactivation groups were also examined for evidence of activation and aneuploidy as described above. In these cases, cumulus‐denuded oocytes were put through the electroactivation chamber but the pulse was not triggered. A further group of oocytes was studied to determine the effect of handling and exposure to hyaluronidase on activation frequency and parthenogenetic pathways. Finally, the spontaneous rate of aneuploidy was examined in fertilised embryos of F1 hybrid female mice × Rb(1.3)1Bnr male mice at first cleavage mitosis.

The results show that single pulse electroactivation does not increase the level of aneuploidy in single‐pronuclear parthenogenones compared to the “sham” group or the spontaneous rate observed in 1‐cell fertilised embryos, nor does aneuploidy appear to increase with postovulatory age. The developmental pathways observed in the electroactivation group are significantly different to those observed in the “sham” group, and the level of activation observed in both groups is increased through handling of oocytes and their exposure to hyaluronidase. © 1993 Wiley‐Liss, Inc.
Separation of bovine X and Y sperm based on surface differences
Authors: Cartwright Elizabeth J. , Harrington Pauline M. , Cowin Allison , Sharpe Paul T.
Published: February 2005

Aqueous two‐phase partition involving thin‐layer counter current distribution (TLCCD) has been used to assess surface heterogeneity of ejaculated bovine sperm. When partitioned in charge‐insensitive aqueous two‐phase systems, which detect non‐charge associated surface properties, the sperm fractionates into two distinct populations. Using a Y‐chromosome‐specific DNA marker, it has been shown that one of these populations is enriched in Y chromosome bearing sperm. However, this population is not pure—it consists of 80% Y sperm, with the other 20% being X sperm. All the sperm in the original population that had begun to undergo the acrosome reaction were separated into this same peak; the sex chromosome composition of these sperm is unknown. Since the aqueous partition of sperm is based on surface properties these results suggest that two populations of Y sperm exist that have different surface characteristics. © 1993 Wiley‐Liss, Inc.
Direct evidence that the mouse sex‐determining gene Sry is expressed in the somatic cells of male fetal gonads and in the germ cell line in the adult testis
Authors: Rossi Pellegrino , Dolci Susanna , Albanesi Cristina , Grimaldi Paola , Geremia Raffaele
Published: February 2005

Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis shows that Sry mRNA is expressed in male fetal urogenital ridges from 12.5 day p.c. embryos, but not in enriched populations of primordial germ cells from the same embryos, indicating that Sry is expressed in the somatic cells of the embryonal gonad at the time of testis determination. We also show that, in the adult male mouse testis, Sry mRNA is expressed at high levels in meiotic and postmeiotic germ cells and, at much lower levels, also in Sertoli cells. Treatment with cyclic adenosine monophosphate (cAMP) analogs of cultured Sertoli cells from postnatal testis completely abolishes Sry mRNA expression. © 1993 Wiley‐Liss, Inc.
Protein secretion by the mouse blastocyst: Stimulatory effect on secretion into the blastocoel by transforming growth factor‐α
Authors: Dardik Alan , Doherty Adam S. , Schultz Richard M.
Published: February 2005

We have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328–333]. In the present study we examine the effect of transforming growth factor‐α (TGF‐α) on these proteins. We observe that TGF‐α stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one‐dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF‐α, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF‐α in mouse preimplantation development. © 1993 Wiley‐Liss, Inc.
Okadaic acid induces premature chromosome condensation reflecting the cell cycle progression in one‐cell stage mouse embryos
Authors: Dyban A. P. , de Sutter P. , Verlinsky Y.
Published: February 2005

Haploid parthenogenetic embryos as well as fertilized mouse eggs were treated in vitro with 1–10 μM okadaic acid (OA) at the one‐cell stage. Cytogenetic analysis detected that OA induces nuclear envelope breakdown (NEBD) and premature condensation of interphase chromosomes in pronuclei as well as in 2nd polar body (PB) nuclei. G1‐, S‐, and G2‐type prematurely condensed chromosomes (PCC) were found in pronuclei of embryos of different age, which reflects their progression through the first cell cycle. In nuclei from 2nd PBs only G1‐ and S‐type PCC were observed. Using the types of PCC as a criterion of different phases of the cell cycle, it was possible to estimate that in haploid parthenogenetic embryos G1‐phase lasts until 5.5 hr post activation (hpa), S‐phase takes from 4.5 to 9.5 hpa, and from 8.5 hpa G2‐phase had started. Second PBs were found to be in G1‐phase until 6.5 hpa and S‐phase started in some as early as 5.5 hpa, but in most not before 7.5 hpa.

Treatment with OA visualizes G1‐chromosomes in pronuclei as well as in 2nd PBs, and it is easy to count the number of these chromosomes and recognize a T6 marker chromosome. The possibility to apply cytogenetic analysis of G1‐chromosomes from 2nd PBs for a more accurate detection of maternal meiotic nondisjunction is discussed. © 1993 Wiley‐Liss, Inc.
Noninvasive chemical enucleation of mouse oocytes
Authors: Fulka Josef , Moor Robert M.
Published: February 2005

A noninvasive method of enucleating mouse oocytes has been developed and evaluated. Strong chromosome to chromosome binding was induced by culturing early metaphase I oocytes in etoposide supplemented medium. Subsequent expulsion of the entire chromosome complex during polar body extrusion was facilitated by exposing the etoposide treated oocytes to a combination of cycloheximide and etoposide during anaphase and telophase. This simple two‐step chemical enucleation procedure yields fully enucleated mouse oocytes in 96% of cases. Chemically enucleated oocytes do not contain maturation promoting factor (MPF) at the end of etoposide‐cycloheximide enucleation. MPF levels are, however, restored during subsequent incubation in drug‐free medium and, after 15 h of post‐enucleation culture, the cytoplasts regain their full capacity for parthenogenetic activation and nuclear remodelling. We believe that this novel enucleation technique will greatly facilitate the research in nuclear transplantation. © 1993 Wiley‐Liss, Inc.
Nuclear transfer in cattle: Effect of nuclear donor cells, cytoplast age, co‐culture, and embryo transfer
Authors: Yang Xiangzhong , Jiang Shie , Farrell Paul , Foote Robert H. , McGrath Ann B.
Published: February 2005

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20–22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8–10 hr or 18–20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co‐culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18–20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen or IVF ambryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology. © 1993 Wiley‐Liss, Inc.
Isolation and biochemical characterization of heparin‐binding proteins from boar seminal plasma: A dual role for spermadhesins in fertilization
Authors: Sanz Libia , Calvete Juan J. , Mann Karlheinz , Gabius Hans‐Joachim , Töpfer‐Petersen Edda
Published: February 2005

Sperm surface‐coated heparin‐binding proteins originating from secretions of the male sexual accessory glands, are known to play a pivotal role as extrinsic regulatory factors during sperm capacitation in many mammalian species. They interact with glycosaminoglycans present in the female genital tract and enhance the subsequent zona pellucida‐induced acrosome reaction. We have isolated heparin‐binding proteins from boar seminal plasma by affinity chromatography on heparin–Sepharose and reverse‐phase HPLC. N‐Terminal sequence analysis of these proteins identified a boar counterpart of the bovine capacitation factors BSP‐A1/2 (also called PDC‐109) and BSP‐A3. Several carbohydrate‐ and zona pellucida‐binding proteins, which belong to the newly described spermadhesin family, were also identified as heparin‐binding proteins. Our results imply that, besides other capacitation factors, members of the spermadhesin family may play a dual role in sperm capacitation and fertilization in the pig. © 1993 Wiley‐Liss, Inc.
Vitelline coat of Unio elongatulus egg: I. Isolation and biochemical characterization
Authors: Focarelli Riccardo , Rosati Floriana
Published: February 2005

In this study we isolated and purified the vitelline coat (vc) of Unio elongatulus eggs in order to investigate its protein and carbohydrate composition. SDS dissolved up to 80% of the vitelline coat protein content whereas 100 mM Ammonimum acetate (AA) at pH 11 and 1 mM lithium diiodosalicylate (LISH) dissolved only 40–50%. The ability of extremes of pH or LIS to solubilize the vitelline coats on eggs was then investigated. The results showed that pH from 7 to 11 progressively dissolved the vitelline coats without gross damage to the oocytes. SDS‐PAGE of the solubilized material revealed only two components corresponding to the main components revealed by SDS‐PAGE of the isolated vcs. These peptides have an apparent MW of 220 and 180 kD, are ConA positive, and seem to be connected to each other to form polycomponents. The latter feature is suggested by the electrophoretic pattern of the solubilized material under nondenaturing conditions. © 1993 Wiley‐Liss, Inc.
Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane
Authors: Harayama Hiroshi , Kusunoki Hiroshi , Kato Seishiro
Published: February 2005

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona‐free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple‐stain technique revealed the percentages of acrosome‐reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley‐Liss, Inc.
Local alteration of cortical actin in Xenopus eggs by the fertilizing sperm
Authors: Chow Robert L. , Elinson Richard P.
Published: February 2005

Rhodamine phalloidin (Rph) staining was used to examine the microfilament organization of the Xenopus laevis egg cortex during the early stages of fertilization. Unactivated eggs possessed a cytochalasin B (CR)‐insensitive Rph‐stained matrix that was reorganized upon egg activation and diminished in the presence of CB. Xenopus laevis sperm caused a temporary local increase in Rph staining on the Xenopus cortex. In CB‐treated eggs, the local increases of cortical Rph staining later changed to a Rph‐free area. These temporary local increases of cortical Rph staining were also observed when Notophthalmus viridescens sperm fertilized Xenopus and Rana pipiens eggs, and were followed by the appearance of concentric rings of stained and unstained areas. Our data suggest that Xenopus and Notophthalmus sperm have activities that can both organize and disrupt the cortical filamentous actin of the Xenopus egg. © 1993 Wiley‐Liss, Inc.
Role of glutathione in the maturation and fertilization of pig oocytes in vitro
Authors: Yoshida Mitsutoshi
Published: February 2005

The present study examined, by treatment of buthionine sulfoximine (BSO), which is a specific inhibitor of glutathione (GSH) synthesis, the role of GSH in the maturation and fertilization of pig oocytes in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughterhouse were cultured for 36 h in Waymouth MB 752/1 with or without BSO (1 mM), fertilized in vitro, and assessed for GSH concentration (before insemination), maturation, and fertilization. The addition of BSO to maturation medium immediately after culture (Group I), 12 h after culture (Group II), or 24 h after culture (Group III) significantly decreased the GSH concentration in pig oocytes compared with the control (P < 0.01), whereas the rate of cumulus mass expansion at 36 h of culture and the rates of nuclear maturation and sperm penetration following in vitro insemination did not differ. However, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the control and Group III than in oocytes matured in Groups I and II (P < 0.01). In experiment 2, when BSO was added to maturation media 15, 18, 21, or 24 h after culture, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the medium supplemented with BSO at 21 or 24 h of culture than in oocytes matured in other groups (P < 0.05 or P < 0.01). The results indicate that GSH synthesis occurs throughout in vitro maturation of pig oocytes and GSH is an important cytoplasmic factor for regulating sperm nuclear decondensation and male pronucleus formation following sperm penetration in pig oocytes. © 1993 Wiley‐Liss, Inc.
Purine control of mouse oocyte maturation: Evidence that nonmetabolized hypoxanthine maintains meiotic arrest
Authors: Downs Stephen M.
Published: February 2005

Hypoxanthine is present in preparations of follicular fluid and has been shown to suppress the spontaneous meiotic maturation of mammalian oocytes in vitro. The present experiments examined the possible role of hypoxanthine metabolism in mediating this meiotic arrest. Four putative inhibitors of the enzyme, hypoxanthine phosphoribosyltransferase (HPRT), which metabolizes hypoxanthine to inosine monophosphate, were tested on lysates of oocyte‐cumulus cell complexes. At a concentration of 1 mM, 6‐mercapto‐9‐(tetrahydro‐2‐furyl)‐purine (MPTF) and 6‐mercaptopurine (6‐MP) suppressed enzymatic activity by 86% and 98%, respectively, while 6‐azauridine and 2,6‐bis‐(hydroxyamino)‐9‐β‐D‐ribofuranosyl‐purine had no effect. MPTF and 6‐MP increased the inhibitory effect of hypoxanthine on germinal vesicle breakdown, but the other agents did not. The 2 active agents had similar effects on salvage activity and hypoxanthine‐maintained meiotic arrest in denuded oocytes. Also, oocytes from XO mice were more sensitive to the meiosis‐arresting action of hypoxanthine than oocytes from XX littermates, which have twice the HPRT activity. The actions of the HPRT inhibitors were not due to their conversion to nucleotides via HPRT and negative feedback on purine de novo synthesis, because azaserine and 6‐methylmercaptopurine riboside, which are more potent inhibitors of de novo synthesis, had a stimulatory, rather than inhibitory, effect on hypoxanthine‐arrested oocytes. Furthermore, several lines of evidence indicate that metabolism of hypoxanthine to xanthine and uric acid by xanthine oxidase does not mediate the inhibitory action of this purine base on meiotic maturation. The data therefore suggest that nonmetabolized hypoxanthine is responsible for the meiotic arrest observed, most likely through suppression of cAMP degradation. © 1993 Wiley‐Liss, Inc.
The sea urchin egg jelly coat is a three‐dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis
Authors: Bonnell Barry S. , Larabell Carolyn , Chandler Douglas E.
Published: February 2005

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a threedimensional network of interconnected fibers extending nearly 50 μm from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a threedimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50–60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick‐freeze/deep‐etch/rotary‐shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix. © 1993 Wiley‐Liss, Inc.
Flow cytometric studies of bicarbonate‐mediated Ca2+ influx in boar sperm populations
Authors: Harrison R. A. P. , Mairet B. , Miller N. G. A.
Published: February 2005

Boar spermatozoa loaded with the Ca2+ probe fluo‐3 were incubated in various Tyrode's‐based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two‐colour flow cytometry; propidium iodide was included in the media to detect membrane‐damaged (“dead”) cells.

If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonatemediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo‐3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it.

The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times. © 1993 Wiley‐Liss, Inc.
ZNC(C)PR affects developmental changes of P46 phosphorylation in rat hippocampus
Authors: Chen Xiu‐Fang , Tang Tong , Zhang Jian‐Wei , Miao Hong‐Hua , Wang Tong‐Xi , Du Yu‐Cang
Published: February 2005

Protein phosphorylation has been suggested to be correlated with brain development and with the molecular mechanism of behavioral effects of neuropeptides. The present study reports in vitro endogenous phosphorylation of P46, a membrane‐associated protein that is changed during development of the rat hippocampus. This study indicated that the degree of endogenous phosphorylation may be correlated with the establishment of synaptic connections. Interestingly, P46 was proved to be identical to a well‐known growth‐associated protein B‐50/GAP‐43 in its identical apparent molecular weight, isoelectric point, phosphorylation dependence, and the cross immunoreaction of monoclonal anti‐B‐50/GAP‐43 antibody and P46. Moreover, neonatal administration of neuropeptide ZNC(G)PR could facilitate the developmental progress of P46 endogenous phosphorylation. It is suggested that the changes in P46 phosphorylation could be involved in the cellular mechanism of ZNC(C)PR behavioral effects on learning. © 1993 Wiley‐Liss, Inc.
Insulin and IGF‐I signaling through the insulin receptor substrate 1
Authors: Keller Susanne R. , Lamphere Lou , Lavan Brian E. , Kuhné Michelle R. , Lienhard Gustav E.
Published: February 2005

The insulin and insulin‐like growth factor‐I (IGF‐I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF‐I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of ˜160 kD, now known as the insulin receptor substrate 1 (IRS‐1). We have purified IRS‐1 from mouse 3T3‐L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS‐1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3‐kinase (PI 3‐kinase) binds tightly to the activated platelet‐derived growth factor (PDGF) and colony‐stimulating factor‐1 (CSF‐1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3‐kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3‐L1 adipocytes, a portion of the Ptyr form of IRS‐1 becomes tightly complexed with PI 3‐kinase. Since IRS‐1 binds to fusion proteins containing the SH2 domains of PI 3‐kinase, association most likely occurs through this domain. The association of IRS‐1 with PI 3‐kinase activates the enzyme about fivefold. Thus, one signaling pathway from the insulin and IGF‐I receptors probably proceeds as follows: tyrosine phosphorylation of IRS‐1, tight association of IRS‐1 with PI 3‐kinase with accompanying activation of the kinase, elevation of the PI 3‐phosphates. © 1993 Wiley‐Liss, Inc.
Gene regulation by IGF‐I
Authors: Baserga Renato
Published: February 2005

Constitutive overexpression of the insulin‐like growth factor‐I (IGF‐I) receptor makes 3T3 cells capable of growing in serum‐free medium supplemented solely with IGF‐I to the total exclusion of the platelet‐derived growth factor (PDGF) and epidermal growth factor (EGF) receptors, the other two receptors that are required for the growth of 3T3 cells. Pietrzkowski et al. (Cell Growth Differ 92:199–205, 1992) found that these cells, overex‐pressing the IGF‐I receptor, can also be induced to grow by the addition of EGF. We have ascertained that the mechanism by which EGF induces growth in these cells is through the induction of substantial amounts of IGF‐I RNA and IGF‐I. In 3T3 cells overexpressing the EGF receptor, a functional IGF‐I receptor is still required for growth. We have also found, using a temperature‐sensitive mutant of the SV40 large T antigen, that the levels of IGF‐I mRNA and of IGF‐I secreted in the medium are markedly increased by a functional T antigen. Using an IGF‐I promoter driving a reporter luciferase gene, we have determined that this increase in the expression of IGF‐I by T‐antigen occurs, at least in part, at a transcriptional level. It seems, therefore, that transformation by SV40 reduces the requirement for growth factors by increasing the production of IGF‐I. © 1993 Wiley‐Liss, Inc.
Parental imprinting of anIGF‐2transgene
Authors: Lee Jacqueline E. , Tantravahi Umadevi , Boyle Ann L. , Efstratiadis Argiris
Published: February 2005

As a consequence of parental imprinting in mice, the paternal allele encoding insulin‐like growth factor‐II (IGF‐II) is expressed, whereas the maternal allele is silent in most tissues. To examine whethercis‐acting sequences involved in imprinting are located in the vicinity of thelgf‐2gene, we have constructed mouse transgenic lines and studied the expression of a 30 kb ratlgf‐2transgene, in which the coding region has been replaced with thelacZreporter sequence. Chromatin position effects and/or absence of long‐range regulatory elements seem to have affected tissue‐specific expression in the transgenic mice. However, in one of six expressing lines, staining of embryos for β‐galactosidase activity was detected in a minor subset of tissues normally transcribing the endogenous homolog, but only when the transgene was transmitted paternally. This transgene was integrated into mouse chromosome 19, which is apparently free of imprinted loci. Although the possibility that thelgf‐2transgene was inserted into an as yet unidentified imprinted iocus is discussed, a more likely interpretation of our results is that the transgene carries at least a portion of its own imprinting signal, because it consists of the genomic sequences of a locus already known to be imprinted and maintains the correct imprinting mode. © 1993 Wiley‐Liss, Inc.
Coexpression of gap junction proteins in the cumulus‐oocyte complex
Authors: Valdimarsson Gunnar , De Sousa Paul A. , Kidder Gerald M.
Published: February 2005

The connexins constitute a family of proteins that make up the intercellular membrane channels of gap junctions. We had previously reported the presence of two members of this protein family, connexins 32 and 43, in mouse one‐cell zygotes (Barron et al., Dev Genet 10:318–323, 1989; Valdimarsson et al., Mol Reprod Dev 30:18–26, 1991), implying that both must be present in the mature oocyte and could be involved in mediating the intercellular coupling that occurs between the oocyte and cumulus granulosa during oogenesis. In the present report we provide evidence for this, based on an analysis of the cumulus‐oocyte complex (COC) using reverse transcription‐polymerase chain reaction (RT‐PCR) and immunocytochemistry with a confocal microscope. Transcripts of both connexin32 (Cx32) and connexin43 (Cx43) were detected by RT‐PCR in both components of the COC. Cx32 mRNA in the oocyte declined precipitously following human chorionic gonadotropin (hCG) stimulation of pregnant mare serum gonadotropin (PMSG)‐primed ovaries, whereas there was no obvious change in Cx43 mRNA. Peptide‐specific antibodies against both connexins provided diffuse cytoplasmic staining of oocytes as well as some punctate staining near the oocyte surface, which could not be unequivocally resolved as cumulus‐oocyte gap junctions. However, the two antibodies did provide clear evidence of Cx32 and Cx43 in gap junction‐like structures between cumulus cells. We could find no evidence of the incorporation of the oocyte's store of Cx32 into gap junctions during postfertilization development. These findings make it very likely that intercellular coupling within the COC involves at least three types of gap junction channels (32–32, 32–43, 43–43), only one of which (43–43) is retained by the preimplantation embryo. © 1993 Wiley‐Liss, Inc.
Growth retardation in rats whose growth hormone gene expression was suppressed by antisense RNA transgene
Authors: Matsumoto Kazuya , Kakidani Hitoshi , Takahashi Akio , Nakagata Naomi , Anzai Masayuki , Matsuzaki Yuichi , Takahashi Yumi , Miyata Kenji , Utsumi Kyozo , Iritani Akira
Published: February 2005

We produced four transgenic founder rats (Fo) by introducing into rat embryos a fusion gene, which consisted of rat growth hormone (GH) promoter containing with four copies of thyroid hormone response element (TRE) and antisense cDNA sequences for rat GH. This transgene promoter directed 2.8‐fold stimulation of CAT gene expression in transfected GH3 rat pituitary tumor cells compared with the rat GH promoter alone. Two of four transgenic rats expressed antisense RNA in the pituitary. Transgenic offspring (F1) from each founder rat exhibited dwarfism at as early as 3–4 weeks of age, and they exhibited ≈︁T70–85% reduced growth rate compared with their nontransgenic littermates over 56 weeks of observation. Plasma rat GH concentration was ≈︁T40–50% lower in transgenic F1 rats compared to their nontransgenic littermates. In these experiments, the pituitary hormone expression controlled in a complex manner was shown to be repressed by the antisense RNA transgene. Furthermore, the suppression of gene expression could be achieved by antisense RNA transgene in the rat as well. © 1993 Wiley‐Liss, Inc.
Spermatogenesis in the black porgy, Acanthopagrus schlegeli (teleostei: Perciformes: Sparidae)
Authors: Gwo J.‐C. , Gwo H.‐H.
Published: February 2005

The ultrastructure of spermatogenesis and of the spermatozoon of Acanthopagrus schlegeli (Sparidae) are described. The testis is of the unrestricted type. Germ cells are surrounded by cyst cells. Spermiogenesis involves conspicuous modifications such as intracellular movements (diplosome and mitochondria migration, nuclear rotation, and depression) and structural changes (chromatin condensation, shape of mitochondria, and loss of cytoplasm). The mature spermatozoon has a spherical nucleus with a deep, axial nuclear fossa, and an unusual notch, shaped like a bow tie. The short midpiece contains four spherical mitochondria and encircles the basal body of the flagellum. It is concluded that the A. schlegeli spermatozoon is of a primitive type, but that it is characterized by a unique feature which may provide a useful systematic character. © 1993 Wiley‐Liss, Inc.
Multiple activation of mouse sperm motility
Authors: Si Yuming , Okuno Makoto
Published: February 2005

To investigate the activation mechanism of mouse sperm motility, the intact sperm in various activities were further investigated after demembranation. When dry sperm was diluted into sucrose solution, the sperm exhibited low motility with the swimming velocity of 13.5 ± 3.8 μm/s and the beat frequency of 1.5 ± 0.4 Hz. The demembranated sperm were immotile in the reactivation solution lacking cAMP. Meanwhile, when dry sperm was diluted into the solution containing either high concentration of NaCl or Ca2+, they exhibited the beat frequency of about 9 Hz. The demembranated ones exhibited the intermediate motility in the absence of cAMP. When dry sperm were diluted into the sucrose solution containing HCO3, the sperm exhibited a vigorous motility with the swimming velocity of 181.2 ± 10.1 μm/s and the beat frequency of 11.3 ± 1.2 Hz. The demembranated sperm exhibited the high reactivation motility (90%) and flagellar beat frequency (9 Hz) in the absence of cAMP. These values were almost equivalent to those obtained in the demembranated sperm pretreated with sucrose or Ca2+ or NaCl and reactivated in the presence of cAMP. The activation induced by bicarbonate was considered complete in comparison with the activation by Ca2+ or NaCl. It was likely that the activation of mouse sperm motility took multiple states. © 1993 Wiley‐Liss, Inc.
Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae
Authors: Giles J. R. , Yang X. , Mark W. , Foote R. H.
Published: February 2005

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley‐Liss, Inc.
Alkaline phosphatase staining of pig and sheep epiblast cells in culture
Authors: Talbot Neil C. , Rexroad Caird E. , Pursel Vernon G. , Powell Anne M.
Published: February 2005

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast‐derived cell cultures were negative for AP staining, with the exception of neuron‐like cultures. Epiblast‐like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm‐endoderm‐like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis. © 1993 Wiley‐Liss, Inc.
Evaluation of mouse sperm acrosomal status and viability by flow cytometry
Authors: Tao Jun , Critser Elizabeth S. , Critser John K.
Published: February 2005

A procedure was developed to evaluate mouse sperm acrosomal status and viability simultaneously utilizing flow cytometry. Four fluorescein isothiocyanate (FITC)‐conjugated lectins, peanut agglutinin (PNA), concanavalin agglutinin (ConA), Pisum sativum agglutinin (PSA), and soybean agglutinin (SBA), were investigated, with PNA providing the greatest sensitivity and specificity in distinguishing acrosome‐present and acrosome‐absent mouse spermatozoa. To expose lectin binding sites, digitonin (20 μM at room temperature for 10 min) was used to permeabilize sperm plasma membranes. Sperm cell viability was determined by Hoechst 33258 (H258) exclusion. To prevent permeabilized cells from staining with H258, salmon sperm DNA (SS‐DNA) was applied to bind excess dye in the solutions after supravital staining. Calcium ionophore (A23187; 5 or 20 μM) was used to induce acrosome reactions. The results of flow cytometric analyses were compared with epifluorescence microscopic observation and were highly correlated (r = 0.999; P < 0.001). The method developed provides an objective and efficient procedure to estimate simultaneously both acrosomal status and viability of mouse spermatozoa. © 1993 Wiley‐Liss, Inc.
Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification
Authors: Rao K. B. C. Appa , Pawshe C. H. , Totey S. M.
Published: February 2005

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y‐chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4‐cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y‐chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y‐chromosome specific primers amplify the similar Y‐chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley‐Liss, Inc.
Presence and possible role of C‐ras and nuclear (c‐fos and c‐jun) proto‐oncogene products in preimplantation embryonic development in mice
Authors: Ahmad Khaliq , Naz Rajesh K.
Published: February 2005

The presence and possible role of products of nuclear (c‐fos and c‐jun) and c‐ras proto‐oncogenes were investigated in preimplantation embryonic development in mice. Polyclonal antibodies to c‐fos or c‐jun proto‐oncogene products did not affect development of in vitro‐cultured embryos from two‐cell to morula or from morula to late blastocyst stages. However, v‐H‐ras monoclonal antibody (mAb) to c‐ras protein (p21), although it did not inhibit the development of in vitro‐cultured embryos from two‐cell to morula stages, it significantly (P < .001–.005) inhibited the development of morula to late blastocyst stages in a dose‐dependent manner. The effects of v‐H‐ras mAb were specific, since immunoabsorption with synthetic ras peptide completely blocked inhibitory effects of v‐H‐ras mAb. Neither c‐fos nor c‐jun antibodies reacted with specific proteins corresponding to c‐fos (62 kDa) and c‐jun (39 kDa) products on the Western blots of various murine ova/embryos extracts. However, the c‐fos and c‐jun antibodies reacted with 62 and 39 kDa protein bands, respectively, on the blot of NIH 3T3 cells extract. The v‐H‐ras mAb specifically identified 21 ± 3 kDa protein corresponding to c‐ras p21 on the blots of early as well as late blastocyst extracts. The rat control ascites IgG1 did not react with any protein band on the blots of various ova/embryo extracts. The reactions of v‐H‐ras mAb on the Western blots of blastocyst extracts were specific, since immunoabsorbed antibody was unable to react with any specific band on blots of early or late blastocyst extract. These results were further confirmed by immunoprecipitation procedure utilizing v‐H‐ras mAb. Again, the v‐H‐ras mAb immunoprecipitated a 21 kDa band from early as well as late blastocyst extracts. The rat control ascites IgG1 did not react with any band corresponding to p 21 in the immunoprecipitation procedure. These results suggest that the specific products of nuclear proto‐oncogenes, the c‐fos and c‐jun, are not detected in murine ova and preimplantation embryos, and the respective antibodies do not inhibit embryogenesis, indicating that they may not play a major role in early embryonic development. On the other hand, the product of c‐ras proto‐oncogene is specifically expressed in the blastocyst‐stage embryos and may have a possible role in preimplantation embryonic development in mice. © 1993 Wiley‐Liss, Inc.
Assessment of ram sperm mitochondrial function by quantitative determination of sperm rhodamine 123 accumulation
Authors: Windsor D. P. , White I. G.
Published: February 2005

A simple procedure is described for determining the functional state of ram sperm mitochondria by quantitative measurement of sperm rhodamine 123 (R 123) accumulation. Sperm were incubated with 1 μg/ml R 123, and the accumulated R 123 was measured fluorimetrically after release from washed sperm by detergent lysis. Ram sperm R 123 uptake was maximal after 30 min of incubation and responded to changes in both sperm (P < 0.01) and R 123 (P < 0.01) concentration. There was a linear relationship (r = 0.98) between R 123 uptake and the proportion of cold‐shocked sperm present in a sperm sample. R 123 uptake was unaffected by 20 mM 2‐deoxyglucose or by 10 mM malonate (the latter being sufficient to reduce O2 uptake; P < 0.01). R 123 accumulation in ram sperm was reduced by 6 mg/ml sodium pentobarbitone (P 0.05), by 1 μM 2,4‐dinitrophenol (P < 0.01), and by 0.05% Triton X‐100 (P < 0.01). It is concluded that quantitative estimation of R 123 uptake complements oxygen uptake in detecting mitochondrial dysfunction in ram sperm. While it is largely unaffected by inhibition of glycolysis, and is less sensitive than oxygen uptake to trichloroacetic acid cycle inhibition, R 123 uptake is sensitive to factors directly reducing the mitochondrial membrane potential of ram sperm. It may therefore be useful in the evaluation of the effects of such membrane‐mediated injuries as cold shock and freezing damage on ram sperm mitochondria. © 1993 Wiley‐Liss, Inc.
Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis
Authors: Regalado Francisco , Esponda Pedro , Nieto Antonio
Published: February 2005

We have compared the biosynthesis of secretory proteins in rabbit cauda epididymidis maintained for 15 days at abdominal temperature with that of the scrotal cauda. Explants from both situations were incubated in vitro in the presence of [35S] methionine, and the labelled proteins released into the incubation medium were analyzed by polyacrylamide gel electrophoresis. Body temperature specifically inhibited the synthesis of at least two polypeptides of 43 kDa and 21 kDa (designated EP21), whereas the synthesis of polypeptides of 80, 39, 31, and 24 kDa was increased. These changes resembled those produced by castration, but androgen treatment was not able to reverse the effect of body temperature. To confirm these observations, poly (A)+ RNA from the scrotal and the abdominal cauda respectively, was translated in vitro and the synthesized products were immunoprecipi‐tated with an antibody against EP21 polypeptide. Both castration and body temperature strongly decreased the concentration of EP21 mRNA. In vivo testosterone administration restored the content of EP21 mRNA in cauda from castrated animals, but not in cauda maintained at body temperature. The changes observed might be related to the adverse effect of body temperature on sperm storage in the cauda epididymidis. © 1993 Wiley‐Liss, Inc.
Temporal pattern of IGF‐I expression during mouse preimplantation embryogenesis
Authors: Doherty Adam S. , Temeles Gretchen L. , Schultz Richard M.
Published: February 2005

Although mouse preimplantation embryos express transcripts for the insulin‐like growth factor‐I receptor (IGF‐IR), IGF‐I transcripts were not detected by reverse transcription‐PCR (RT‐PCR) at any stage of preimplantation development in a previous study (Rappolee et al., Genes Dev 6:939–952, 1992). We report that IGF‐I transcripts are detected in the preimplantation embryo by RT‐PCR and describe the temporal pattern of expression of this transcript from the oocyte to blastocyst stages; the level declines from the oocyte to 8‐cell embryo and then increases from the 8‐cell to blastocyst stages. An explanation is offered to account for the differences in detecting the IGF‐I transcript, and the results are discussed in the context of an autocrine/paracrine IGF‐I circuit in the preimplantation mouse embryo. © 1994 Wiley‐Liss, Inc.
Stage‐specific appearance of the mouse antigen TEC‐3 in normal and nuclear transfer bovine embryos: Re‐expression after nuclear transfer
Authors: van Stekelenburg‐Hamers Annelies E. P. , Rebel Heggert G. , van Inzen Wouter G. , de Loos Frans A. M. , Drost Maarten , Mummery Christine L. , Weima Sjerp M. , Trounson Alan O.
Published: February 2005

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC‐3 by indirect immunofluorescence. During preimplantation embryo development TEC‐3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32‐cell stage. The finding that TEC‐3 is not expressed before the onset of embryonic transcription, which occurs at the eight‐cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC‐03 antibody showed that the TEC‐3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program. © 1994 Wiley‐Liss, Inc.