bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of RU486 on development and implantation of rat embryos
Authors: Roblero Luis S. , Croxatto Horacio B.
Published: February 2005

This study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1+2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose‐related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486‐treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486‐treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486‐treated pseudopregnant recipients failed to implant completely when the dose was ≥ 1.0 mg.

The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.
Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis
Authors: Srivastava Archana , Olson Gary E.
Published: February 2005

To investigate surface glycoprotein changes during post‐testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS‐PAGE fractionated samples using biotinylated lectins and Vectastain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation‐dependent sperm surface glycoproteins co‐migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation‐related surface changes occurring within the epididymis than are noted with surface labeling procedures.
Stored mRNA in early embryos of a fern Marsilea vestita: A paternal and maternal origin
Authors: Kuligowski Janine , Ferrand Michèle , Chenou Eliane
Published: February 2005

In early embryos of the fern Marsilea vestita, we have shown that transcription did not begin until the 8‐cell stage, even though protein synthesis was taking place. As in animal species, the pre‐existence of stored maternal mRNA has been suspected in embryonic cells. To investigate the presence of such long‐lived mRNA we have used in situ hybridization method using [3H]polyuridylic acid, [3H]poly(U), as a probe.

Temporal and spatial changes in the distribution of poly(A)+ RNA were followed throughout all the different phases of embryogenesis. Poly(A)+ RNA was present in the cytoplasm of both unfertilized and fertilized eggs and early embryos, while the nucleus exhibited no, or a moderate, [3H]‐poly(U) binding activity. New poly(A)+ RNA was detected in the nucleus only at the 16‐cell stage, when cytological differentiation of the root apical cell became morphologically detectable.

After in situ hydridization with the labeled probes, the numerous silver grains detected autoradiographically over the mature sperm, indicate the presence of poly(A)+ RNA molecules associated with the male genome.

We discuss the possible role of poly(A)+ RNA of maternal origin in supporting early translation prior to synthesis of new mRNA. Additional studies are needed to elucidate the role of stored paternal mRNA.
Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo
Authors: Cohen Jacques , Feldberg Dov
Published: February 2005

The present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure‐eight shape. Seventy‐two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P < 0.001) more AT‐blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT‐embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD‐embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.
Quantitative light and scanning electron microscopy of ferret sperm
Authors: Van Der Horst Gerhard , Curry Patrick T. , Kitchin Robert M. , Burgess Warren , Thorne E. Tom , Kwiatkowski Don , Parker Michael , Atherton Robert W.
Published: February 2005

Sperm were obtained via electro‐ejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black‐footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black‐footed ferret.
Qualitative and quantitative changes in protein sythesis of bovine follicular cells during the preovulatory period
Authors: Rabahi Flora , Monniaux Danielle , Pisselet Claudine , Chupin Daniel , Durand Philippe
Published: February 2005

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H‐methionine or with 35S‐methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the prevoulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH‐stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.
Improvement of fertilizing performance by normal and abnormal mouse semen after zona opening of mature oocytes
Authors: Barlow P. , Van Der Zwalmen P. , Schoysman R. , Leroy F.
Published: February 2005

The usefulness of opening the zona pellucida by partial zona dissection (PZD) to enhance fertilization of mature mouse oocytes was studied after insemination with three types of semen: normal and diluted semen and semen from long‐term‐vasectomized males. Zona opening did not by itself induce parthenogenetic cleavage of mature oocytes and did not significantly increase mono‐ and polyspermic fertilization of oocytes inseminated with normal semen. While a fertilization rate of 62% was obtained among intact oocytes, of which 4.5% were polyspermic, a 66.8% fertilization rate was observed among PZD oocytes, 6.3% of which were polyspermic. However, after using diluted semen, only 54 of 193 intact oocytes were fertilized (28%), and PZD improved the fertilization rate to 65.4%. Cleavage rate of nonmanipulated oocytes inseminated with abnormal semen from vasectomized males was dramatically decreased in comparison with those inseminated with normal semen (7.6% vs. 65%). PZD induced a moderate but significant improvement of fertilization performance when using this abnormal semen (19.6%).
Time course and pattern of cortical granule breakdown in hamster eggs after sperm fusion
Authors: Stewart‐Savage J. , Bavister Barry D.
Published: February 2005

We have determined the temporal relationship between sperm fusion and cortical granule breakdown in the hamster egg. Sperm fusion was determined by the Hoechst‐transfer method (Stewart‐Savage and Bavister: Dev Biol 128:150–157, 1988), and cortical granules were visualized with fluorescein isothiocynate‐conjugated Lens culinaris agglutinin (Cherr et al. J Exp Zool 246:81–93, 1988). By 55 min after insemination, there was an 85% reduction in the density of cortical granules (fewer than four granules/100 m̈m2). Taking this value as the completion of the cortical reaction, analysis of the data indicate that the cortical reaction was completed 9 min after sperm fusion and 3 min after the formation of the zona and cell surface blocks to polyspermy. There was no obvious spatial pattern of granule loss in eggs that had a Hoechst‐positive sperm but had not completed the cortical reaction.
Ontogeny of Ha‐ras and c‐myc mRNA levels in rabbit embryo and extraembryonic tissues by quantitative in situ hybridization
Authors: Martinoli M. G. , Lambert R. D. , Pothier F. , Pelletier Georges
Published: February 2005

A large variety of proto‐oncogenes are known to be of key importance in cellular growth and differentiation during embryonic development. Using quantitative during in situ hybridization, we studied in detail the levels of the proto‐oncogenes Ha‐ras and c‐myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prental life of rabbit. cDNA probes encoding for Ha‐ras (fragment Kpn 1‐BstE II of 883 bp) and c‐myc (fragment Pst 1‐Pst 1 of 490 bp) were used to detect specific transcripts in fixed cryostat sections. High levels of Ha‐ras and c‐myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha‐ras and c‐myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha‐ras and c‐myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto‐oncogenes, Ha‐ras and c‐myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto‐oncogenes Ha‐ras and c‐myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prental development.
Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation
Authors: Rochwerger Leonora , Cuasnicu Patricia S.
Published: February 2005

Rat epididymal glycoprotein DE (37kDa) associates with the sperm surface during maturation and is localized over the dorsal region of the acrosome. In the present study we examine, by indirect immunofluorescence, the localization of DE after in virto and in vivo capacitation. While 49% of sperm capacitated in vitro for 5 hr still presented fluorescence over the dorsal region, 51% showed labeling distributed over a domain that corresponds to the equatorial segment of the sperm head. This change in the localization of fluorescence was not associated with sperm deterioration or death and increased gradually as a function of capacitation time, reaching the maximum at 5 hr. The presence of labeling over the equatorial segment results from protein migration and cannot be induced by permeabilization, proteinase, or high ionic strength treatments. The omission of Ca2+ from the standard capacitation medium inhibitied the relocalization of DE, and incubation with Ca2+ ionophore A23187 for induction of the acrosome reaction (AR) significantly raised the percentage of cells with DE localized over the equatorial region. Finally, while free and cumulus‐associated spermatozoa recovered from the oviducts of in vivo inseminated females presented 15% and 21% of cells with redistribution respectively, all perivitelline (acrosome reacted) spermatozoa showed DE over the equatorial segment. These results indicate that epididymal protein DE migrates to the equatorial segment under in vitro and in vivo capacitating conditions and suggest a possible association between the redistribution of DE and the occurrence of the AR.
Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae
Authors: Vasiliev Aleksej O. , Kapkov Denis V. , Kandror Konstantin V. , Stepanov Aleksandr S.
Published: February 2005

A homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained. By means of immunoblotting, enzyme‐linked immunosorbent assay (ELISA), and immunochemical isolation on protein A‐Sepharose, it is shown that the amount of casein kinase 2 increases under heat shock conditions (at least in part due to the synthesis de novo), while the synthesis of the majority of other proteins falls. The activity of casein kinase 2 is supressed during heat shock and so does not correlate with its content. The results give an evidence for the two‐step model of casein kinase 2 regulation during heat shock.
The main adenosine triphosphate‐binding component of amphibian oocytes is ferritin
Authors: Kandror Konstantin V. , Stepanov Aleksandr S. , Tsuprun Vladimir L.
Published: February 2005

After fractionation of mitochondrion‐free extracts of Xenopus laevis and Rana temporaria oocytes in sucrose gradients, a distinct peak of adenosine triphosphate (ATP)/guanosine triphosphate (GTP)‐binding activity in the 50–70 S range has been detected. This substance has a boyant density in Cs2SO4 of 1.45 g/cm3. The nucleotide‐binding substance has been purified to apparent homogenety. By means of electron microscopy, sodium dodecyl sulfate‐electrophoresis and other methods it has been identified as ferritin.
Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles
Authors: Pavlok A. , Lucas‐Hahn A. , Niemann H.
Published: February 2005

This study investigated the capacity of healthy oocytes derived from follicles of different size to undergo normal fertilization and early embryonic development in vitro and full‐term development in vivo. Ovaries were collected from a local abattoir and dissected and classified as follows: group A, >4–8 mm (large); group B, >2–4 mm (medium); and group C, >1–2 mm (small). Oocytes were isolated by puncturing the follicular wall and pressing of the follicle. Only healthy‐looking cumulusoocyte complexes (COC) were used for in vitro maturation. Oocytes were fertilized in vitro by frozen/thawed semen from one bull. Approximately one‐fourth of all oocytes was fixed and stained 15–20 h after fertilization, to determine penetration rates. The remaining eggs were transferred to culture medium and were cultivated for up to 9.5 days. Cleavage was observed 65 h and 7 days after fertilization. Expanded, hatching, and hatched blastocysts were fixed and stained after 9.5 days of culture. A total of 86 blastocysts derived from group A and B oocytes was nonsurgically transferred to synchronized recipients 7–8 days after onset of culture. A total of 6.624 follicles were dissected from 265 ovaries, and 1,485 oocytes were isolated from 1,671 group A follicles, 3,509 oocytes from 3,862 group B follicles, and 965 oocytes from 1,091 group C follicles. The fertilization rate, rate of normal fertilization, rate of polyspermy, and rate of other abnormal fertilization features were as follows: group A, 84.9%, 43.2%, 34.1%, 7.6%; group B, 83.6%, 44.8%, 31.1%, 7.8%; and group C, 61.7%, 13.1%, 33.7%, 19.1%. The cleavage rate and the percentage of embryos with 5–8 cells after 65 h amounted in group A to 68.9% and 36.9%; in group B, 64.3% and 32.7%; and in group C, 19.7% and 0.4%, respectively. The percentage of blastocysts on days 7 and 9.5 as well as the rate of hatched blastocysts were 27.7%, 24.8%, and 21.7%, respectively, in group A; 20.9%, 19.2%, and 16.8%, respectively, in group B; and zero in group C. Transfer of 38 blastocysts of group A to 19 recipients led to 10 pregnancies; transfer of 48 blastocysts to 24 recipients of group B resulted in 12 pregnancies. It is concluded that blastocysts from follicles ranging from >2 to 8 mm in size have a similar developmental potential. Oocytes from 1‐ to 2‐mm follicles, however, have a significantly lower competence to undergo in vitro maturation and fertilization and, under the conditions of our experiments, completely lack the capability to cleave beyond the 8‐cell stage.
Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo
Authors: Watson Andrew J. , Hogan Aileen , Hahnel Ann , Schultz Gilbert A. , Wiemer Klaus E.
Published: February 2005

The sensitive technique of mRNA phenotyping with the reverse transcription‐polymerase chain reaction was employed to determine the patterns of gene expression for several growth factor ligand and receptor genes during bovine preimplantation development. Several thousand bovine embryos encompassing a developmental series from one‐cell zygotes to hatched blastocysts were produced by the application of in vitro maturation, fertilization, and oviductal epithelial cell embryo coculture methods. Transcripts for transforming growth factor (TGF‐α) and platelet‐derived growth factor (PDGF‐A) are detectable in all preimplantation bovine stages as observed in the mouse. Transcripts for TGF‐β2 and insulin‐like growth factor (IGF‐II) and the receptors for PDGF‐α, insulin, IGF‐I, and IGF‐II are also detectable throughout bovine preimplantation development, suggesting that these mRNAs are products of both the maternal and the embryonic genomes in the cow, whereas in the mouse they are present only following the activation of the embryonic genome at the two‐cell stage. In contrast to the mouse embryo, IGF‐I mRNA was detected within preimplantation bovine embryos. Basic fibroblast growth factor (bFGF) is a maternal message in the bovine embryo, since it is only detectable up until the eight‐cell embryo stage. Bovine trophoblast protein (bTP) mRNA was detectable within day 8 bovine blastocysts. As was observed in the mouse, the transcripts for insulin, epidermal growth factor (EGF), or nerve growth factor (NGF) were not detectable in any bovine embryo stage. Analyses of this type should aid the development of a completely defined culture medium for the more efficient production of preimplantation bovine embryos.
Sea urchin small RNA ribonucleoprotein particles: Identification, synthesis, and subcellular localization during early embryonic development
Authors: LeBlanc Janine M. , Infante Anthony A.
Published: February 2005

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross‐linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8–12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartment, newly synthesized small RNAs have a predominantly cytoplasmic localization. All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.
Cyanide‐resistant reduction of nitroblue tetrazolium and hydrogen peroxide production by the rabbit blastocyst
Authors: Manes Cole
Published: February 2005

The ability of the rabbit blastocyst to reduce nitroblue tetrazolium (NBT) to formazan in the presence of cyanide was assayed as an indicator of extramitochondrial oxidase activity capable of generating the superoxide radical. A cytochemical method initially developed for the detection and localization of hydrogen peroxide production at the ultrastructural level in phagocytosing leukocytes (Briggs et al.: J Cell Biol 67:566, 1975) was also applied to the blastocyst. The results demonstrate that the rabbit blastocyst acquires the ability to reduce NBT by a cyanide‐insensitive process and to generate hydrogen peroxide between the fourth and fifth days postcoitum. The enzymatic activity responsible is apparently an NAD(P)Hdependent oxidase in the outer, microvillous plasma membrane of the trophoblast.
Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro
Authors: Younis Abdelmoneim I. , Brackett Benjamin G.
Published: February 2005

Efforts to improve bovine embryonic development in vitro involved study of effects of thyroid stimulating hormone (TSH) alone or in combination with LH on bovine oocyte maturation (IVM). Putative effects were assessed by observing cumulus expansion (CE), fertilization (IVF), and development to morulae/blastocysts (M/B). Effects of prolactin (PRL) were also investigated. Variables for the 24‐hr IVM interval were no hormone (control), TSH (0.1, 0.5, or 1.0 μg/ml) or PRL (10, 100, or 1000 μg/ml), luteinizing hormone (LH) (0, 10, or 100 μg/ml) + TSH (0.1 or 0.5 μg/ml), and serum (20%, v/v) + 0.5 μg TSH/ml; data were from 4–5 trials for each IVM treatment. Higher proportions of oocytes exhibited complete CE with hormones or serum than without (P < 0.05). All oocytes (with and without CE) were inseminated with heparin‐capacitated sperm. A higher proportion of inseminated oocytes cleaved after IVM with 0.5 μg TSH/ml (53.4%) than for other TSH treatments (P < 0.05). The combination of TSH (0.1 and 0.5 μg/ml) with 10 μgLH/ml for IVM enabled higher proportions (P < 0.05) of ova to fertilize (67.4 and 69.2%) than did medium alone (28.3%), LH (10 μg/ml) alone (54.1%), or serum + 0.5 μg TSH/ml (55.6%). No improvement in proportions undergoing fertilization was seen after addition of TSH to 100 μgLH/ml for IVM. Frequency of CE and cleavage did not differ among PRL treatments. More M/B developed from cleaved ova after IVM with LH or TSH than with PRL or no hormone (P < 0.05). Data document the beneficial influences of TSH and LH during IVM on the quality of the resulting ova.
Highly acetylated H4 is associated with histone displacement in rat spermatids
Authors: Meistrich Marvin L. , Trostle‐Weige Patricia K. , Lin Rueyling , Allis C. David , Bhatnagar Yogendra M.
Published: February 2005

The presence of highly acetylated histone H4 during spermatogenesis was studied to evaluate its correlation with the events of gene transcription, histone deposition, and histone displacement. We utilized an antibody raised to a pentaacetylated synthetic peptide that preferentially recognizes highly (tetra‐ and tri‐) acetylated forms of rat testis H4. Electrophoretic separation of histones from enriched fractions of spermatogenic cells followed by detection of these forms by staining and by immunoblotting using this antibody showed that the highly acetylated forms were limited almost exclusively to spermatids beginning at step 11 of development. Immunofluorescence generally confirmed this result but also detected a high degree of acetylation in steps 9 and 10. Immunofluorescence also revealed a striking polarity in the progression of histone from the spermatid nucleus. Highly acetylated H4 was displaced from the anterior to the caudal portion of the spermatid nucleus during steps 11 and 12, along with other histones, prior to their displacement by transition proteins. Thus, while monoacetylated and low levels of diacetylated forms of H4 were associated with stages at which histone deposition and transcription occur, the more highly acetylated forms appeared in high levels only at the stage at which histone displacement occurs.
Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt‐stored zona pellucida penetration assay
Authors: Andrews J. C. , Howard J. G. , Bavister B. D. , Wildt D. E.
Published: February 2005

The ability of domestic cat or leopard cat spermatozoa to penetrate zonae pellucidae (ZP) of salt‐stored, domestic cat oocytes was examined as an assay for sperm capacitation. Ovarian oocytes were recovered after ovariectomy and matured in vitro for 18–36 h. Following removal of cumulus cells, the oocytes were used fresh, or stored (4°C, 0.5‐24 weeks) in a HEPES‐buffered hypertonic salt solution. Electroejaculated, washed sperm (2‐4 × 106 sperm/ml) were preincubated for 1.0 h (38°C, 5% CO2 in air) and then co‐incubated (2 × 105 sperm/ml) with fresh or stored oocytes for 6.0 h. Gametes were incubated in a protein‐free, modified Tyrode's solution (TLP‐PVA) or in the same medium containing 4.0 mg/ml bovine serum albumin (BSA; TALP‐PVA). Treatments were compared for percentage ZP penetration (defined as sperm heads reaching more than halfway through the ZP) as an index of sperm capacitation. In both the domestic cat and leopard cat, there was no difference (P > 0.05) in sperm penetration of fresh ZP (domestic cat, 42.5 ± 5.4%; leopard cat, 38.6 ± 2.8%) or stored ZP (domestic cat, 32.4 ± 4.2%; leopard cat, 27.6 ± 2.3%). Sperm incubated in protein‐free medium (TLP‐PVA) were less capable (P<0.05) of ZP penetration (domestic cat, 14.6 ± 5.9%; leopard cat, 7.9 ± 3.0%) than sperm incubated in medium TALP‐PVA containing BSA (domestic cat, 60.3 ± 5.9%; leopard cat, 58.4 ± 3.0%). These data indicate that (1) albumin facilitates capacitation and ZP penetrating ability of cat spermatozoa; (2) domestic cat ZP appear to lack a block to heterospecific penetration by “foreign” (leopard cat) sperm; and (3) penetration of stored domestic cat ZP can be used as an index of sperm capacitation in the domestic cat and the leopard cat.
Changes in protein synthesis pattern during in vitro maturation of goat oocytes
Authors: Le Gal F. , Gall L. , De Smedt V.
Published: February 2005

The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide‐containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis. © 1992 Wiley‐Liss, Inc.
Assessment of Pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa
Authors: Farlin M. E. , Jasko D. J. , Graham J. K. , Squires E. L.
Published: February 2005

The use of fluorescein‐conjugated Pisum sativum agglutinin (FITC‐PSA) was evaluated for its ability to distinguish acrosome‐intact from acrosome‐damaged stallion spermatozoa. Incubation of fresh (acrosomeintact) and frozen‐thawed (acrosome‐damaged) spermatozoa with FITC‐PSA resulted in acrosome‐intact spermatozoa that exhibited no fluorescence, while acrosome‐damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome‐intact and acrosome‐damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozenthawed spermatozoa in a sample) of FITC‐PSA binding. The binding of FITC‐PSA increased in samples as the portion of frozen‐thawed (acrosome‐damaged) to fresh (acrosomeintact) spermatozoa increased. A positive correlation existed (r = 0.98, P < 0.05) between the percentage of FITC‐PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome‐damaged spermatozoa, determined by electron microscopy using gold‐conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC‐PSA binding may be useful in determining acrosomal integrity of fresh and frozen‐thawed stallion spermatozoa. © 1992 Wiley‐Liss, Inc.
Release of tissue‐type plasminogen activator by activated rat eggs and its possible role in the zona reaction
Authors: Zhang X. , Rutledge J. , Khamsi F. , Armstrong D. T.
Published: February 2005

The resumption of meiosis results in synthesis of tissue‐type plasminogen activator (tPA) in the rat and mouse oocytes (Haurte et al., Cell 43:551–558, 1985). The present study demonstrates that freshly ovulated rat oocytes released their tPA into the surrounding medium upon in vitro activation by sperm penetration or treatment with a calcium ionophore. The presence of a neutralizing monoclonal anti‐tPA antibody during in vitro activation by the calcium ionophore inhibited the activation‐induced zona hardening and also preserved the ability of the oocyte to be penetrated by sperm subsequent to activation. Rat oocytes undergo zona hardening during in vitro maturation in the absence of serum, presumably as a result of spontaneous cortical granule release, based on findings in mice and hamsters. In the present study, the anti‐tPA antibody prevented the zona hardening and enhanced partition by spermatozoa of rat oocytes that were matured in vitro without serum. Collectively, the observations reported here suggest a possible role of tPA released during the cortical granule reaction in the zona reaction, which contributes to the block to polyspermy.
The role of TGF‐βs in mammalian development and neoplasia
Authors: Akhurst Rosemary J. , Fitzpatrick David R. , Fowlis Deborah J. , Gatherer Derek , Millan Fergus A. , Slager Hans
Published: February 2005

To date, three mammalian TGF‐β isoforms have been identified, each encoded by different genetic loci. Through each is very similar in primary amino acid structure, there are clear differences both in the mature bioactive peptide region and in the latency‐associated peptide, which could potentially confer differential biological specificity.

As one route to investigate differential biological function in vivo we have used gene specific probes for in situ hybridization studies to examine the distribution of RNA transcripts during mammalian embryogenesis. Mouse embryos from 6 to 14.5 gestational age and human embryos from 32 to 57 days post‐fertilization have been probed. A general conclusion from these studies is that each TGFβ gene has a distinct, through overlapping, pattern of transcript distribution and that this pattern, in most cases, is conserved between mouse and man. We have focused on the biological function the TGF‐betas play in certain epithelia and in cardiogenesis, which will be discussed in this presentation. © 1992 Wiley‐Liss, Inc.
The role of growth factors in embryonic induction in Xenopus laevis
Authors: Dawid Igor B. , Taira Masanori , Good Peter J. , Rebagliati Michael R.
Published: February 2005

Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor‐β (TGF‐β) family. TGF‐β2 and TGF‐β3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF‐β named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.

How do inducing factors affect the differentiation of the responsive tissue? One approach to this question has been to look for gene expression in response to induction, especially the activation of regulatory loci like homeobox genes. Several homeobox‐containing genes including Mix.1, Xhox3, X1Hbox1, and X1Hbox6, goosecoid and members of a new class of genes named Xlim, are activated by inducing factors with different patterns of expression in the embryo. Differential expression of regulatory genes probably controls the formation of distinct tissues in an orderly pattern during embryogenesis. © 1992 Wiley‐Liss, Inc.
The bone morphogenetic protein family and osteogenesis
Authors: Wozney John M.
Published: February 2005

The BMPs (bone morphogenetic proteins) are a group of related proteins originally identified by their presence in bone‐inductive extracts of demineralized bone. By molecular cloning, at least six related members of this family have been identified and are called BMP‐2 through BMP‐7. These molecules are part of the TGF‐beta superfamily, based on primary amino acid sequence homology, including the absolute conservation of seven cysteine residues between the TGF‐betas and the BMPs. The BMPs can be divided into subgroups with BMP‐2 and BMP‐4 being 92% identical, and BMP‐5, BMP‐6, and BMP‐7 being an average of about 90% identical. To examine the individual activities of these molecules, we are producing each BMP in a mammalian expression system. In this system, each BMP is synthesized as a precursor peptide, which is glycosylated, processed to the mature peptide, and secreted as a homodimer. These reagents have been used to demonstrate that single molecules, such as BMP‐2, are capable of inducing the formation of new cartilage and bone when implanted ectopically in a rodent assay system. Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research. Based on the chondrogenic and osteogenic abilities of the BMPs in the adult animal, the expression of the mRNAs for the BMPs has been examined in the development of the embryonic skeleton by in situ hybridization. These studies demonstrate that the BMP mRNAs are spatially and temporally expressed appropriately for the proteins involved in the induction and development of cartilage and bone in the embryonic limb bud. Furthermore, primary preparations of limb bud cells respond to BMP‐2, as do several cell lines of the osteoblastic lineage. In addition to expression in the skeletal system, various of the BMP mRNAs are expressed in distinct tissues, suggesting additional roles during development. © 1992 Wiley‐Liss, Inc.
Zygotic gene activation in the mouse embryo: Involvement of cyclic adenosine monophosphate‐dependent protein kinase and appearance of an AP‐1‐like activity
Authors: Schwartz Daniel A. , Schultz Richard M.
Published: February 2005

Protein phosphorylation catalyzed by the cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (PKA) is implicated in regulating zygotic gene activation in the two‐cell mouse embryo (Poueymirou and Schultz; Dev Biol 133:588–599, 1989). We now provide evidence that H8, which is a PKA inhibitor, inhibits expression of an hsp70‐driven β‐galactosidase reporter gene and that the concentration‐dependence of this inhibition is similar to that for inhibiting expression of a stage‐specific gene(s) that is a product of zygotic gene activation. We also demonstrate that neither cAMP nor serum can stimulate the expression, as detected by a histochemical assay, of a cAMP response element (CRE)‐ or serum response element (SRE)‐driven β‐galatosidase reporter gene, respectively in either germinal vesicle‐intact oocytes or aphidicolin‐arrested one‐cell embryos that are chronologically at the tw‐cell stage. In contrast, although 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) does not stimulate expression of a TPA response element (TRE)‐driven β‐galatosidase reporter gene in germinal vesicle‐intact oocytes, it stimulates such expression in aphidicolin‐arrested one‐cell embryos. Moreover, TPA can stimulate the expression of either a CRE‐ or an SRE‐driven β‐galatosidase reporter gene in such embryos. Results of these studies further implicate protein phosphorylation in regulating zygotic gene activation, along with its role in modulating enhancer function in the early mouse embryo.
Golden hamster embryonic genome activation occurs at the two‐cell stage: Correlation with major developmental changes
Authors: Seshagiri Polani B. , McKenzie Debbie I. , Bavister Barry D. , Williamson Judy L. , Aiken Judd M.
Published: February 2005

The earliest time of onset of embryonic genome activation in golden hamsters was investigated. The inhibition of transcription by α‐amanitin (11 μg/ml) in cultured embryos resulted in a total arrest of development of early 2‐cell embryos (26 hr post‐egg activation); under similar conditions, immediate cleavage divisions of 1‐, late 2‐, 4‐, and 8‐cell embryos were not affected. Electrophoretic analysis of [35S]methionine‐labeled embryonic proteins showed that α‐amanitin treatment apparently inhibited transcription‐dependent protein synthesis in early 2‐cell and, to some extent, in late 2‐cell when compared to 4‐cell embryos. Analysis of total RNA synthesis, using [α32P]‐UTP or [32P]‐orthophosphate, showed that there was a high proportion of radioactivity associated with the macromolecular fraction (RNA) at the early and late 2‐cell stages and at the 4‐cell stage compared to that at the 1‐cell stage. These results indicate that the de novo synthesis of RNA, encoded by the embryonic genome, occurs at the 2‐cell stage and that the second and subsequent cleavage divisions of hamster preimplantation embryos are dependent on new transcriptional activity. This initial activity of the embryonic genome in hamsters is coincident with several characteristic features of in vitro development such as a block to development, synthesis of major proteins, change in energy substrate preference, phosphate‐inhibition of development and a requirement for amino acids.
Primary culture of human fallopian tube epithelial cells and co‐culture of early mouse pre‐embryos
Authors: Takeuchi Kazuhiro , Nagata Yukihiro , Sandow Bruce A. , Hodgen Gary D.
Published: February 2005

We have established a monolayer culture system for human fallopian tube epithelial cells. The cells were isolated from tubes using collagenase digestion, and were cultured in Ham's F‐10 supplemented with 15% fetal calf serum. The epithelial cells derived from culture were characterized using immunocytochemical staining and electron microscopy. These cells were stained with antikeratin and anti‐epithelial membrane antigen, but showed no staining after treatment with antivimentin. Electron microscopy showed many microvilli on the cell surface and tight junctions or desmosomes at areas of cell‐cell contact. Cell proliferation was enhanced by epidermal growth factor, but not by fibroblast growth factor, insulin, transferrin, estradiol‐17β, or progesterone.

The 2‐cell ICR mouse pre‐embryos were co‐cultured for 4 days with tubal epithelial cells (A) (n = 98), in cell‐conditioned medium (B) (n = 83), or in medium alone (C) (n = 72). During the first 24 h in culture, for groups A and B, the rates of cleavage to the 4‐cell stage were 90.9% and 81.9%, respectively. Cleavage rates in these two groups were significantly higher (P =0.0012, P > 0.00001) than in group C (56.9%). After 72 h in culture, the rates of development to the blastocyst stage were significantly higher for groups A and B compared to group C (89.6% and 73.5% vs. 54.5%, P > 0.00001, P = 0.0002). These results suggest that factor(s) from tubal epithelial cells may facilitate the development of mouse pre‐embryos throughout the pre‐implantation stages.
Cleavage rate of diandric triploid mouse embryos during the preimplantation period
Authors: Henery C. C. , Kaufman M. H.
Published: February 2005

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one‐cell‐stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used (1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, (2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and (3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted. The cell doubling time of the diandric triploid embryos was 13.55 hr (± 1.25), and this was not significantly different from the various controls. The cell doubling time of (1) the micromanipulated controls was 12.12 hr (± 1.16), (2) the control embyros incubated in cytoskeletal inhibitors 10.87 hr (± 0.75), and (3) the group that was briefly incubated in tissue culture 12.43 hr (± 0.74). There was no significant effect of manipulation or incubation in cytoskeletal inhibitors on cleavage rate. Our findings indicate that triploid embryos divide at the same rate as diploid embryos during the preimplantation period.