bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro
Authors: Fukui Y.
Published: February 2005

The role of follicle cells in the acrosome reaction of frozen‐thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus‐oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona‐ and cumulus‐enclosed oocytes. The proportion of live, acrosome‐reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome‐reacted spermatozoa that inseminated cumulus‐enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3% respectively). The frequency of in vitro fertilization was significantly higher for cumulus‐enclosed oocytes (65.4%) than for denuded and corona‐enclosed oocytes (30.8% and 39.4%, respectively). Cumulus‐enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two‐ to eight‐cell, 59.8%; eight‐cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona‐enclosed oocytes. No eight‐cell embryos or more advanced stages of embryonic development were observed in either denuded or corona‐enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.
Time‐Lapse analysis and normal stages of development of cleavage and blastocyst formation in the marsupials the brown antechinus and the stripe‐faced dunnart
Authors: Selwood Lynne , Smith David
Published: February 2005

The first six normal stages of celavage and blastocyst formation in vitro of the brown antechinus and the stripe‐faced dunnart are described based on 429 embryos from 37 dunnarts and 143 embryos from 32 antechinus and on information from previous studies. Embryos were cultured in Dulbecco's modified Eagle's medium with high glucose and 10% foetal calf serum at 35oC and 5% CO2 in air. Seven antechinus and 18 dunnart embryos were cultured in Rose chambers and filmed by time‐lapse cinematography, with an exposure of 0.2 sec and at a rate of 1 frame/30 sec. A rim of zona was formed around the yolk mass during early cleavage. Blastomeres attached to this rim, ensuring that the zona in the hemisphere containing the yolk mass was the first to be lined by blastomeres during blastocyst formation, which is completed at about the 32‐cell stage. Two foci were established during cleavage. The descendants of the first cell to divide at the four‐cell stage included, at both the fourth and fifth division, the first cell to divide and the cell with the shortest cell cycle time and had the shortest average cell cycle time at each division. The descendants of the cell opposite the first cell to divide at the four‐cell stage included, at both the fourth and fifth divisions, the last cell to divide and the cell with the longest cell cycle time and had the longest average cell cycle time at each division.
Ca2+ uptake during capacitation of mouse spermatozoa and the effect of an anion transport inhibitor on Ca2+ uptake
Authors: Ruknudin A. , Silver I. A.
Published: February 2005

With a specially constructed chamber, Ca2+ uptake by mouse spermatozoa was monitored continuously during capacitation and the acrosome reaction. It was shown, using calcium ion‐selective microelectrodes, that there was an initial uptake of Ca2+ by spermatozoa undergoing capacitation. Such net transport was also promoted by the divalent cation ionophores A23187 or ionomycin. An anion inhibitor, SITS, produced dose‐dependent inhibition of Ca2+ uptake. This inhibitor reduced the incidence of capacitation as revealed by a reduction in the B pattern by chlortetracycline (CTC) assay and thus inhibited fertilization, suggesting that anions are involved in calcium uptake in mouse spermatozoa.
Effect of equilibration period on the viability of frozen‐thawed mouse morulae after rapid freezing
Authors: Takahashi Yoshiyuki , Kanagawa Hiroshi
Published: February 2005

Mouse morulae were frozen with 1.5–4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5–30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P < 0.05) were observed when equilibration periods were extended for 3–5 min at 3.0 and 4.0 M and for 5–10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerollactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol‐lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.
Activation of cumulus‐free mouse oocytes
Authors: Marcus G. J.
Published: February 2005

Attempts were made to activate parthenogenetically oocytes from randombred mice under conditions required for nuclear transfer, viz. in the absence of cumulus cells. Of the large number of stimulants previously described and presumed to be generally effective, only exposure to strontium ion (as a replacement for calcium ion in the medium), or to calcium ionophore, activated a substantial proportion of cumulus‐free oocytes from TRf mice, a synthetic, random‐bred strain. These stimuli were less effective in oocytes from random‐bred ICR mice. The relevance of the findings to nuclear‐transfer cloning attempts in mice is discussed.
Ejaculate‐hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona‐free hamster ova and zona‐intact domestic cat oocytes
Authors: Howard Jogayle , Wildt David E.
Published: February 2005

Electroejaculate traits and circulating follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona‐free hamster ova and zonaintact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim‐up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim‐up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P< 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P< 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P< 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P< 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P< 0.05) by the NS compared to the DR and SU treatments. This study (1) provides baseline ejaculate and endocrine norms for the leopard cat, (2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona‐intact domestic cat oocytes, (3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovam penetration, and (4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.
Regulation of mouse preimplantation development: Inhibitory effect of the calmodulin antagonist W‐7 on the first cleavage
Authors: Poueymirou William T. , Schultz Richard M.
Published: February 2005

The calmodulin antagonist W‐7 inhibits cleavage of 1‐cell mouse embryos in a concentration‐dependent manner. This inhibition is likely to be specific for a calmodulin‐mediated process, since the less active congener W‐5 does not inhibit cleavage when used at concentrations of W‐7 that do. Concentrations of W‐7 that inhibit cleavage and do not inhibit either the uptake or incorporation of [35S]methionine do inhibit [3H]thymidine incorporation; similar concentrations of W‐5 do not inhibit [3H]thymidine incorporation. Consistent with W‐7′s ability to inhibit cleavage by inhibiting DNA synthesis is that addition of W‐7 at later times that correspond with exit from S phase results in cleavage to the 2‐cell stage. Although W‐7 does inhibit cleavage of 1‐cell embryos, it does not inhibit transcriptional activation, which occurs in the 2‐cell embryo and is characterized by the synthesis of a group of proteins of Mr = 70,000. Results of these experiments suggest a role for calmodulin in the first cell cycle of the mouse embryo and provide another example in which zygotic gene activation is not dependent on progression through the first cell cycle.
Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus)
Authors: Nakanishi Akira , Utsumi Kyozo , Iritani Akira
Published: February 2005

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 106 − 107 spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4′,6′‐diamidino‐2‐phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1–3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.
Identification of heparin‐binding proteins in bovine seminal plasma
Authors: Chandonnet L. , Roberts K. D. , Chapdelaine A. , Manjunath P.
Published: February 2005

A group of four similar proteins, BSP‐A1, BSP‐A2, BSP‐A3, and BSP‐30‐kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP‐A1, ‐A2, ‐A3, and ‐30‐kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin–Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin–Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well‐characterized BSP proteins indicated the presence of BSP‐A1, BSP‐A2, BSP‐A3, and BSP‐30‐kDa proteins. These results demonstrate that BSP‐A1, ‐A2, ‐A3, and ‐30‐kDa proteins bind to heparin or glycosaminoglycans and we postulate interaction of these spermatozoa‐bound proteins with glycosaminoglycans in the female reproductive tract.
Immunocytochemical studies of hamster oocyte activation
Authors: Sinosich M. J. , Lanzendorf S. E. , Hodgen G. D.
Published: February 2005

By indirect immunofluorescence, using rabbit anti‐heparin‐binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically and non‐physiologically (microsurgically) activated hamster gametes. Whereas mature gametes (sperm, metaphase II oocytes) were negative, in vivo conceived preimplantation embryos, from pronuclear to two‐ and four‐cell stages, were HBPP positive. No HBPP was demonstrated in the zona pellucida, but HBPP‐dependent immunofluorescence was localized in the perivitelline space. Oocytes incubated with hyaluronidase demonstrated variable responses from negative to positive. (Diluent or sperm) microinjected oocytes were all activated and HBPP positive within 4 h after stimulation. Thus neither activation by microinjection nor HBPP expression required paternal gametes. These kinetics suggest that HBPP may be a cortical granule secretogogue which can be applied to monitor oocyte responses during in vitro manipulations.
Injection of isolated centriolar complex to induce mitosis in starfish eggs
Authors: Yamada Hisafumi , Kuraishi Ritsu , Amikura Reiko M. , Nagano Hiroshi
Published: February 2005

When centriolar complex isolated from starfish spermatozoa was injected into starfish immature oocytes (fully grown, germinal vesicle stage) followed by treating the latter with 1‐methyladenine, mitotic events such as condensation and division of chromosomes of the female pronucleus and cytokinesis following completion of meiosis were observed. No cortical reaction detected in the oocytes. Essentially the same was noted for mature oocytes (pronuclear stage) into which the centriolar complex had been injected. The oocytes that had received sperm tail fraction or buffer alone did not initiate cleavage. It would thus appear that sperm centriolar complex is significantly essential to the initiation of cleavage.
Expression of genes for insulin and insulin‐like growth factors and receptors in early postimplantation mouse embryos and embryonal carcinoma cells
Authors: Telford Nancy A. , Hogan Aileen , Franz Carolyne R. , Schultz Gilbert A.
Published: February 2005

The expression of genes for insulin and insulin‐like growth factors (IGFs) and their receptors was examined in early postimplantation mouse embryos and differentiating F9 embryonal carcinoma cells using mRNA phenotyping. Messenger RNA phenotyping involves the reverse transcription of RNA followed by amplification of specific target cDNA sequences using the polymerase chain reaction (PCR). The identities of the resulting PCR fragments were confirmed using at least two of the following methods: (1) size determination by agarose gel electrophoresis, (2) the presence of diagnostic restriction sites, (3) hybridization with radio‐labeled cDNA probes, (4) sequencing of the PCR fragment. Transcripts for insulin receptors, IGF‐I receptors, and IGF‐II receptors were detected in RNA samples from day 7.5 to day 9.5 mouse embryos and in F9 cells, although the level of insulin receptor mRNA in F9 cells was very low. Transcripts for both IGF‐I and IGF‐II ligands were also detectable in the embryo and F9 RNA samples, but transcripts for insulin ligand were undetectable in either set of material. The results suggest that insulin does not act as a paracrine or autocrine growth factor in early postimplantation embryos or F9 cells but that both embryos and F9 cells have the potential to respond to exogenous (e.g., maternal) sources of insulin. Both IGF‐I and IGF‐II could act as paracrine or autocrine growth factors, and IGF‐II is the more abundant growth factor in differentiating F9 cells.
Expression of the insulin‐like and platelet‐derived growth factor genes in human uterine tissues
Authors: Boehm Keith D. , Daimon Makoto , Gorodeski Israel G. , Sheean Leon A. , Utian Wulf H. , Ilan Judith
Published: February 2005

The human uterus repeatedly exhibits cyclic biochemical and cytological changes during the reproductive period of life. These changes are the result of a well‐characterized endocrine network involving the hypothalamus, pituitary, and ovary. The exact nature of the mechanism(s) by which the sex steroids act on the uterus remains to be elucidated. Possible local mediators of hormonal action on the uterus include polypeptide growth factors. Using the method of RNA transfer blot hybridization, we have analyzed tissue samples from the cycling human endometrium and tissue samples of human myometrium and myometrial benign tumor (leiomyoma) for the presence of platelet‐derived growth factor (PDGF) and insulin‐like growth factor (IGF) RNA. All the uterine tissues examined possessed RNA for PDGF‐B chain and IGF‐I and ‐II. Two transcripts were observed for PDGF‐B chain, four were observed for IGF‐I, and eight were observed for IGF‐II. Overall, the relative abundance of PDGF‐B chain RNA was consistent in all of the uterine tissues examined. In contrast, IGF RNA relative abundance varied. IGF‐I RNA was highest in late proliferative stage endometrium, and IGF‐II RNA was highest in early proliferative stage endometrium. Both IGF‐I and IGF‐II RNAs were greater in amount in leiomyoma than in myometrium. The increased IGF‐I RNA in late proliferative–stage human endometrium correlates with the known elevation of estradiol secretion by the ovary and the increased concentration of uterine estradiol receptors during this stage of the menstrual cycle. Furthermore, these data are consistent with reports of increased IGF‐I RNA in the rat uterus in response to administration of estradiol. Elevated levels of IGF RNA in leiomyomas may be related to the genesis and/or progression of these myometrial tumors.
Assessment of the viability and acrosome status of fresh and frozen‐thawed human spermatozoa using single‐wavelength fluorescence microscopy
Authors: Centola G. M. , Mattox J. H. , Burde S. , Leary J. F.
Published: February 2005

The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen‐thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC‐conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome‐intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI‐neg/PSA‐neg—viable, physiologic acrosome reacted (AR); group II, PI‐neg/PSA–pos—viable, non‐AR; group III, PI‐pos/PSA‐neg–‐nonviable, non‐AR; group IV, PI‐pos/PSA–neg—nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen‐thawed sperm may undergo premature break‐down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.
Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey
Authors: Morgan Patricia M. , Boatman Dorothy E. , Bavister Barry D.
Published: February 2005

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30–32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty‐six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty‐five embryos developed to the six‐ to eight‐cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six‐ to eight‐cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.
Localization of bindin expression during sea urchin spermatogenesis
Authors: Nishioka David , Ward Rita D. , Poccia Dominic , Kostacos Connie , Minor Joseph E.
Published: February 2005

Expression of the bindin gene was examined in testicular cells of the sea urchin Strongylocentrotus purpuratus. In situ hybridization studies, using an 35S‐labeled antisense RNA probe transcribed from a bindin cDNA, reveal that bindin mRNAs are localized in spermatogenic cells displaced towards the lumens of maturing testicular acini. Little or no hybridization is observed in spermatogenic cells displaced towards the perivisceral epithelium or in somatic cells of the testis. A similar localization of the bindin protein itself is observed using a rhodamine‐conjugated polyclonal antibody against bindin, which shows a punctate immunofluorescence pattern in late spermatogenic cells. Immunogold labeling of ultrathin sections and electron microscopy reveal that this punctate immunofluorescence is an apparent result of localized deposits of bindin in intracellular vesicles. Through the terminal stages of spermatogenesis, these bindin‐containing vesicles apparently fuse to form the single acrosomal vesicle of the mature spermatozoon. These results indicate (1) that bindin mRNAs are transcribed relatively late in spermatogenesis, (2) that bindin is translated soon after production of its mRNA, (3) that bindin quickly associates with intracellular vesicles during or soon after its synthesis, and (4) that these vesicles fuse to form the single acrosomal vesicle during the terminal stage of spermatogenesis.
Regulation of laminin gene expression in the expansion of mouse blastocysts
Authors: Kang Hae Mook , Kim Kyungjin , Kwon Hyuk Bang , Cho Wan Kyoo
Published: February 2005

This study aims to elucidate gene expression of laminin and its role in expansion of the blastocyst during mouse early embryogenesis. The gene expression of laminin, in particular the B1 subunit and the synthesis of laminin polypeptides, was examined during the expansion of blastocyst by a RNA‐blot hybridization with 32P‐labeled laminin B1 cDNA and immunoprecipitation followed by a SDS‐PAGE, respectively. Laminin B1 transcript was actively expressed in the blastocyst stage of embryos. The gene expression of laminin B1 and the synthesis of laminin protein were also increased when blastocyst was expanded. Treatments of cAMP analogue, isobutylmethylxanthine, forskolin, and cholera toxin, which are known to stimulate the blastocyst expansion, increased laminin B1 transcript levels and synthesis of laminin polypeptides. Treatment with retinoic acid, a known regulator of laminin gene expression, not only increased the gene expression of laminin but stimulated the blastocoel expansion without a significant increase in intracellular cAMP levels.

These results indicate that laminin gene expression may play an important role in the process of blastocyst expansion in the mouse preimplantation embryos.
Development of preimplantation rabbit embryos in uterine flushing–supplemented culture media
Authors: Fischer Bernd , Jung Thomas , Hegele‐Hartung Christa , Beier Henning M.
Published: February 2005

The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F‐10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared. The uterine flushings derived from donors of 0.5–6 years of age. Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration. Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation). The growth retardation was more pronounced in blastocyts (recovered at day 4 post coitum [p.c.]) than in morulae (recovered at day 3 p.c.). Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development. Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages. Advanced donor ages, heating up to 80°C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos. The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium.
Combined effects of protein synthesis and phosphorylation inhibitors on maturation of mouse oocytes in vitro
Authors: Motlík Jan , Rimkevičová Zora
Published: February 2005

In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 μg/ml), followed by further 3 hours in cycloheximide only, lowered the rate of GVBD (93% and 92%, respectively). It means that 3 and 5 hours preincubation in cycloheximide did not impair the ability of mouse oocytes to resume meiosis in medium with the protein synthesis inhibitor. To test the combined effects of inhibition of protein phosphorylation and protein synthesis, oocytes were cultured for 3, 4, or 5 hours in 2 mM of 6‐DMAP and subsequently for 3 hours in 10 μg/ml cycloheximide. The incubation in 6‐DMAP for 4 or 5 hours diminished (63% or 35% of GVBD, respectively) the ability of mouse oocytes to resume meiosis when subsequent protein synthesis was blocked by cycloheximide. However, the highly condensed bivalents were always visible in GVs. Thus the above treatment did not prevent chromatin condensation although GVBD was blocked.
Protein synthesis inhibitors prevent both spontaneous and hormone‐dependent maturation of isolated mouse oocytes
Authors: Downs Stephen M.
Published: February 2005

The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor‐free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methyl‐xanthine (IBMX), or delayed by follicle‐stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone‐induced maturation. CEO were maintained in meiotic arrest for 21–22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle‐stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose‐dependent fashion. This was accompanied by a dose‐dependent suppression of 3H‐leucine incorporation by oocyte‐cumulus cell complexes. The action of these inhibitors on FSH‐ and epidermal growth factor (EGF)‐induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH‐treated groups, below even that of the control (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF‐treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP. In a time course experiment, it was determined that CX could prevent FSH‐induced maturation if added as late as 3 h after the onset of culture, but the drug was less effective in EGF‐treated cultures. It is concluded that a protein with a rapid turnover rate is involved in the spontaneous maturation of mouse oocytes and that de novo protein synthesis is a requirement for hormone induction of GVB in vitro.
Decondensation of mouse sperm chromatin in cell‐free extracts: A micromethod
Authors: Maleszewski Marek
Published: February 2005

A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensation in vitro using very small volumes of cytoplasmic preparations, even smaller than 1μl. We show that cell‐free extracts obtained from interphase HeLa cells as well as lysates from mouse eggs and embryos can sustain early stages of mouse sperm nucleus transformation, provided the sperm nuclear envelope is damaged or removed.
Characterization of human alpha fetoprotein charge microheterogeneity during fetal development
Authors: Keel Brooks A. , Harms Robin L. , Leal Juan A. , Cho Sechin
Published: February 2005

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by‐products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5‐4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl<4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15–20‐week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23–26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with >95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, >97% of the AFP bound to conA. In contrast, when AFP isoforms IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.
Culturing two‐ to eight‐cell caprine embryos using domestic chicken eggs
Authors: Blakewood E. G. , Pool S. H. , Prichard J. F. , Godke R. A.
Published: February 2005

Early‐stage caprine embryos were placed in the chick embryo amnion to determine if this culture method would support the development of embryos from a farm animal species. Following superovulation and natural mating, two‐ to eight‐cell embryos were surgically collected from crossbred donor goats. Embryos were allotted to in vitro culture treatments across two different experiments (EXP). In EXP‐I, embryos allotted to Treatment A (control) were cultured in Ham's F‐10 with 10% fetal calf serum and 1% antibiotic‐antimycotic (HF‐10). Embryos in Treatment B were placed on a bovine fetal uterine fibroblast monolayer in HF‐10, embryos allotted to Treatment C were agarose embedded and injected into the amniotic cavity of a day‐4 chick embryo and those placed in Treatment D were co‐cultured in HF‐10 with day‐15 caprine trophoblastic vesicles. In EXP II Treatments A, B, and C were the same; however Treatment D was omitted. EXP‐I and EXP‐II also differed in that chick embryo co‐culture was for 72 hr in EXP‐I but was extended to 96 hours in EXP‐II. Additionally, the monolayer co‐culture was limited to 96 hr in EXP‐II; whereas, embryos in EXP‐I remained on monolayer culture for 96 hr plus an additional 72 hr for subsequent embryo evaluation. Results indicate that the amniotic cavity of the developing chick embryo enhanced the development of two‐ to eight‐cell caprine embryos through to hatching blastocysts when compared with that of the control medium alone. In addition, the beneficial effects of monolayer co‐culture appeared to be lost when the caprine embryos were removed from the monolayer, whereas, the in vitro viability of these embryos cultured in the chick embryo amnion was maintained after removal from the amniotic cavity.
Production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae
Authors: Picard L. , Chartrain I. , King W. A. , Betteridge K. J.
Published: February 2005

Bovine inner cell masses (ICMs) were isolated by immunosurgery at day 8 or 10, or by dissection at day 14, and combined with day‐5.5 morulae. Aggregation was obtained between 89%, 62%, and 0% of the day‐5:day‐8, day‐5:day‐10, day‐5:day‐14 composites, respectively. Chromosome analysis of composites potentially carrying the 1/29 translocation as a chromosome marker and temporarily transferred to the bovine uterus for 8 days showed that chimaeric day‐14 embryos can be obtained from day‐5:day‐8 aggregation. The definitive transfer of eight day‐5:day‐8 and 11 day‐5:day‐10 composites resulted in the birth of six and four calves, respectively; five of the six, but none of the four, were chimaeric. The five chimaeras showed mostly the ICM phenotype. The morphological differences between ICMs at different stages of development were examined by electron microscopy and related to the success of the aggregation technique. It is concluded that bovine embryonic cells can regulate for at least 3 days difference in development but not 5 days even though aggregation is still possible.
Zona drilling enhances fertilization by mouse caput epididymal sperm
Authors: Wazzan Wassim C. , Gwatkin Ralph B. L. , Thomas Jr. Anthony J.
Published: February 2005

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500–1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P=0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.
Observations of hamster sperm‐egg fusion in freeze‐fracture replicas including the use of filipin as a sterol marker
Authors: Clark Judy M. , Koehler James K.
Published: February 2005

We have extended the observations of previous transmission electron microscopy studies of sperm‐egg fusion to include those of freeze‐fracture replicas showing sperm‐egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5–30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds β‐OH‐sterols to form visible complexes in freeze‐fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome‐reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm‐egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin‐rich egg membrane and the filipin‐poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin‐sterol complexes are not found in sperm‐derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.
Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70
Authors: Hendrey Jaqueline , Kola Ismail
Published: February 2005

Eukaryotic and prokaryotic cells have been shown to respond to physical and chemical stress by the induction of proteins called heat shock proteins. Heat shock protein 70 (Hsp70), is the most ubiquitous of these proteins. Although heat shock proteins are generally thought to protect cells from physiologically stressful stimuli, it cannot be assumed that this is so, because several cases exist in which thermotolerance is acquired without the production of heat shock proteins, and in several other cases the hyperproduction of these heat shock proteins does not produce thermotolerance. In this study we show that unfertilized mouse oocytes are sensitive to elevated temperatures, and that the synthesis of Hsp70 cannot be induced in these oocytes. Furthermore, our data demonstrate that the expression of Hsp70 in mouse oocytes is sufficient for the acquisition of thermotolerance. Mouse oocytes were injected with mRNA for Hsp70, and the viability of these oocytes was determined after heating. The number of viable oocytes was significantly higher in the group injected with Hsp70 mRNA and then heated compared with oocytes injected with Hsp70 antisense mRNA and sham‐injected controls treated in an identical manner. No significant differences in the number of viable oocytes were found between the group that had been injected with Hsp70 mRNA, heated, and then allowed to recover for 3 hr and the group maintained at 37°C throughout. This study demonstrates that the injection of Hsp70 mRNA into unfertilized mouse oocytes significantly enhances their thermotolerance, indicating that the thermolability of mouse oocytes is due to the lack of expression of Hsp70. This study provides direct evidence for the role of Hsp70 in the enhancement of thermotolerance of mammalian cells.
The spermicidal effect of ethylene dibromide in bulls and rams
Authors: Amir David
Published: February 2005

The spermicidal effect of ethylene dibromide (EDB) in bulls and rams is reviewed. Following oral or parenteral administration EDB was found to cause, by its alkylating effect in the testes of treated bulls and rams, lysis of the chromatin of the elogating spermatids during the time of somatin histones replacement by the sperm protamines. However, while in the bulls the abnormal spermatozoa issued from the affected spermatids were also collected in the ejaculates, this was not the case with treated rams. In the latter animals the abnormal spermatids seem to be phagocytized in the epididymis before their arrival in the ejaculate. In addition, whereas the alkylating effect of EDB occurred also in the upper parts of the epididymis of the bulls, causing tail and acrosome defects to the spermatozoa, in the rams such an effect seems to occur all along the epididymal duct. These differences between bulls and rams in the sites of the genital tract where the drug takes effect, and in the mechanism of this effect, reveal probable differences in the physiology of the reproductive tract between these species.
Structure and expression of the polyubiquitin gene in sea urchin embryos
Authors: Gong Zhiyuan , Cserjesi Peter , Wessel Gary M. , Brandhorst Bruce P.
Published: February 2005

A cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3′ noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock‐inducible during embryogenesis.
Water insoluble fraction of egg yolk maintains porcine sperm motility by activating adenylate cyclase
Authors: Okamura N. , Onoe S. , Sugita Y. , Paquignon M. , Dacheux F. , Dacheux J.‐L.
Published: February 2005

The decrease in motility of porcine cauda epididymal sperm was less than that of caput epididymal sperm in the medium containing bicarbonate. This may be due to the difference of sensitivity of adenylate cyclase to bicarbonate between mature and immature sperm; activation of mature sperm enzyme by bicarbonate was higher than that of immature sperm. Nondialysable fraction of egg yolk prevented the decrease in motility of immature sperm in the presence of bicarbonate, but it was not effective for the motility of mature sperm under the same condition, because only bicarbonate is sufficient for the maintenance of its motility. In the absence of bicarbonate, both mature and immature sperm required egg yolk to maintain motility.

The favorable effect of egg yolk on the motility is ascribed to the enhancement of intracellular cAMP level. Partial fractionation of egg yolk showed that water‐insoluble lipoprotein fraction contains factor(s) which activates adenylate cyclase in sperm plasma membrane. This is the first report in which high molecular weight activator of the sperm enzyme was demonstrated.
The vitelline coat spikes: A new peculiar structure of Mytilus galloprovincialis eggs with a role in sperm‐egg interaction
Authors: Focarelli Riccardo , Rosa Domenico , Rosati Floriana
Published: February 2005

In the course of this study we found that in Mytilus galloprovincialis eggs long filamentous protrusions never described before, which we have termed “vitelline coat spikes,” could be clearly detected using the lectin from Dolichos biflorus, which recognizes the GalNAc residues. The spikes could be also observed by transmission electron microscope but only in some fortuitous sections could their origin in the vitelline coat be clearly observed. The spikes were also clearly visible using the scanning electron microscope. Observations of the sperm‐egg interaction very few seconds after insemination or using fixed eggs suggested that the spikes could play a role in a primary binding to the unreacted sperm. Experiments have been done to test the effect of GalNAc on the sperm‐egg binding and on the fertilization process which seem to confirm this hypothesis.
Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? cytochemical studies
Authors: Royere D. , Hamamah S. , Nicolle J. C. , Lansac J.
Published: February 2005

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen‐DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P < 0.01). Feulgen‐DNA content significantly increased after capacitation in either B2 or follicular fluid (P < 0.05, P < 0.001 respectively), and so did sperm nuclear surface area (P < 0.001). In this study we observed a negative correlation between Feulgen‐DNA content and fertilization rate (P < 0.02). Moreover, the greater effects on Feulgen‐DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P < 0.05) and Feulgen‐DNA content higher (P < 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.