Development of embryos produced by intracytoplasmic sperm injection of cat oocytes

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.     

Yeast infection of sperm, oocytes and embryos after intravaginal culture for embryo transfer

We report two patients treated with interferon-alpha who developed Raynaud's phenomenon followed by multiple digital necrosis. Arteriography of the legs revealed diffuse distal narrowing. Histological examination of the resected tissue showed ischaemic necrosis within the dermis and subcutaneous tissue without vasculitis. Resolution of the symptoms was observed when interferon-alpha was withdrawn. Since other causes of Raynaud's phenomenon and digital necrosis were ruled out, a drug cause is likely. Raynaud's phenomenon should be looked for in patients treated with interferon-alpha. If present, immediate withdrawal of the drug is required.     

The effects of lithium on neurulation stage mouse embryos

The aim of this study was to evaluate the maternal toxicity and teratogenicity of lithium following intraperitoneal injection (i.p.) with lithium carbonate (Li2CO3) in pregnant CD-1 mice at the developmental stage of neurulation (E8; day of vaginal plug, E0). Light (LM) and electron (TEM) microscopic studies were also done to document the tissue and cellular changes occurring in embryonic tissues during the 48 h following treatment with 300 mg/kg body wt. Li2CO3. Controls were untreated or given equimolar amounts of NaCl or Na2CO3. A pharmacokinetic study showed that lithium was rapidly absorbed from the peritoneal cavity after the above-stated dose, achieved peak serum levels of 9.8 mmol/l within 1 h, had a half-life in the blood of 5 h and was completely cleared by 16 to 24 h after injection. Doses of Li2CO3 > 300 mg/kg body wt. were toxic to adult CD-1 mice. The latter dose had no detectable maternal toxicity but caused a 19% resorption rate and 2% incidence of open cranial neural tube defect in gestations terminated on E18. The malformation and resorption rates in gestations terminated on E11, E12 and E14 were not significantly different from those of E18. A strong litter effect was seen both for the resorption and malformation rates at all stages examined. At 3 h after treatment cell death became evident in the neuroepithelium. Cells continued to die for approximately 17 h and all necrotic debris had been cleared by 48 h. Also at 3 h after treatment small densely stained inclusions began to appear in mesodermal cells. TEM showed these to be non-membrane bound with an irregular shape and variable size; the lack of staining for acid phosphatase indicated a non-lysosomal structure; the ultrastructural features suggested a lipoid basis. At 24 h after treatment vascular ruptures and surface ectodermal ruptures were seen in the cranial mesoderm. These ruptures with extravascated blood were also seen at 48 h after treatment. A litter effect was also noted with respect to the tissue and cellular changes. These experiments suggest that the developing vascular system may be a target for lithium. In addition, the possibility is discussed that lithium induced cell death in the neuroepithelium may lead to neural tube defects.     

Post-implantation mouse embryos have the capability to generate and release reactive oxygen species

The capability of the mouse embryo to generate reactive oxygen species (ROS) was examined. Post-implantation embryos were carefully harvested on Day 8 of pregnancy and the production of ROS was quantified using luminol-sensitized chemiluminescence. The embryos were stimulated with either phorbol myristate acetate (PMA) or all-trans-retinal (retinal) and the reaction kinetics were followed over 10 min. ROS secretion was directly proportional to the number of embryos and was suppressed 56% by superoxide dismutase (SOD), 25% by mannitol and as little as 16% by catalase. Embryos deprived of trophoblast showed no light emission suggesting that the source of ROS generation is the trophoblast. Dihydronicotinamide adenine dinucleotide (NADH)-dependent oxidase activity in the plasma membrane of the trophoblast surface was demonstrated by cytochemical methods. The release of ROS into the extracellular medium during the phagocytic process has been related to the cytolytic effect exhibited by these molecules and, perhaps by this means, the trophoblast can play an active role in the phagocytosis of maternal cells during the process of embryo implantation.     

Developmental and dysmorphogenic effects of glufosinate ammonium on mouse embryos in culture

The effects of glufosinate ammonium on embryonic development in mice were examined using whole embryo and micromass cultures of midbrain and limb bud cells. In day 8 embryos cultured for 48 hr, glufosinate caused significant overall embryonic growth retardation and increased embryolethality to 37.5% at 10 micrograms/ml (5.0 x 10(-5) M). All embryos in the treated groups exhibited specific morphological defects including hypoplasia of the prosencephalon (forebrain) (100%) and visceral arches (100%). In day 10 embryos cultured for 24 hr, glufosinate significantly reduced the crown-rump length and the number of somite pairs, and produced a high incidence of morphological defects (84.6%) at 10 micrograms/ml. These embryos were characterized by blister in the lateral head (100%), hypoplasia of prosencephalon (57.1%), and cleft lips (42.9%) at 20 micrograms/ml (10.0 x 10(-5) M). Histological examination of the treated embryos showed numerous cell death (pyknotic debris) present throughout the neuroepithelium in the brain vesicle and neural tube, but did not involve the underlying mesenchyme. In micromass culture, glufosinate inhibited the differentiation of midbrain cells in day 12 embryos with 50% inhibition occurring at 0.55 microgram/ml (2.8 x 10(-6) M). The ratios of 50% inhibition concentration for cell proliferation to cell differentiation in limb bud cells were 0.76 and 1.52 in day 11 and 12 embryos, respectively. These findings indicate that glufosinate ammonium is embryotoxic in vitro. In addition to causing growth retardation, glufosinate specifically affected the neuroepithelium of the brain vesicle and neural tube, leading to neuroepithelial cell death.     

Polypeptide profiles of human oocytes and preimplantation embryos

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.     

Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos

Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation. LIF is also expressed by the extraembryonic membranes of the early mouse embryo. Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development. Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05). LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro. LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005). These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth. In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female. Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture. This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones. These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation. Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis.     

Micromanipulation and cloning studies on buffalo oocytes and embryos using nucleus transfer

1. Nine male volunteers were exposed to the pyrethroid insecticide cyfluthrin. The study was performed in an exposure room, where an aerosol containing cyfluthrin was sprayed to obtain atmospheres with mean cyfluthrin concentrations of 160 and 40 micrograms/m3. Four volunteers were exposed for 10, 30 and 60 min at 160 micrograms/m3 and another five volunteers were exposed for 60 min at 40 micrograms/m3. For 160 micrograms/m3 exposure urine samples were collected before and immediately after exposure as well as for the periods 1-2, 2-3, 3-4, 4-5, 5-6, 6-12 and 12-24 h after exposure. For 40 micrograms/m3 exposure urine samples were collected before and 2 h after exposure. 2. The main urinary cyfluthrin metabolites, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylycyclopropane carboxylic acid (DCCA) and 4-fluoro-3-phenoxybenzoic acid (FPBA), were determined. The limit of detection (LOD) for all metabolites was 0.0025 microgram in an urine sample of 5 ml (0.5 microgram/l). After inhalative exposure of 40 micrograms cyfluthrin/m3 air for 60 min, the amount of metabolites in urine collected in the first 2 h after exposure was less than the LOD, namely 0.14 microgram for cis-DCCA, 0.15-0.28 microgram for trans-DCCA and 0.12-0.23 microgram for FPBA. 3. Of the metabolites, 93% was excreted within the first 24 h (peak excretion rates between 0.5 and 3 h) after inhalative exposure of 160 micrograms/m3. The mean half-lives were 6.9 h for cis-DCCA, 6.2 h for trans-DCCA and 5.3 h for FPBA. 4. The mean trans-:cis-DCCA ratio was 1.9 for the time course as well as for each subject. 5. The amount of metabolites in urine depends on the applied dose, on the exposure time and shows interindividual differences.     

Human cadaver skin viability for in vitro percutaneous absorption: storage and detrimental effects of heat-separation and freezing

To evaluate the clinical results of circulatory support for severe heart failure after operation, we examined 62 patients (39 males and 23 females) who underwent circulatory support for postoperative heart failure from 1984 to 1996. Their ages ranged from 22 to 78 (mean 52) years. In 62 patients, 35 had valvular, 25 had ischemic, and 2 had congenital heart disease. Postoperation, 29 patients underwent venoarterial bypass (VAB), 20 had biventricular bypass (BVB), and 8 had left ventricular bypass (LVB). The remaining 5 patients received a pulsatile left ventricular assist device (LVAD). The weaning and discharge rates of the patients by type of support were 51.7% and 31.0% with VAB, 75.0% and 55.0% with BVB, 87.5% and 37.5% with LVB, and 60.0% and 40.0% with LVAD, respectively. The complete results of this series (64.5% weaning rate and 40.3% discharge rate) were acceptable.     

Meiotic and developmental competence of mouse antral oocytes

Mouse antral oocytes show two different patterns of chromatin organization, defining oocytes with or without chromatin surrounding the nucleolus (SN: surrounded nucleus; NSN: not surrounded nucleus). We have previously shown that upon injection of eCG, NSN antral oocytes shift towards the SN kind of chromatin organization. We hypothesized that these newly formed SN oocytes were those that would have been ovulated after an ovulatory stimulus. The main objective of this study was to investigate the meiotic and developmental competence of these two types of oocytes. SN and NSN antral oocytes were isolated after i.p. administration of eCG + hCG or eCG-only, in vitro-cultured until completion of metaphase II, and inseminated with capacitated spermatozoa; and their development to the 4-cell stage was examined. This study demonstrates 1) that SN and NSN oocytes isolated after injection of eCG + hCG are capable of embryonic development, but not beyond the 2-cell stage; and 2) that SN and NSN oocytes isolated after injection of eCG-only are capable of developing to the 2-cell stage, but a significantly higher number (11.9%) of SN oocytes than NSN oocytes (1.5%) reach the 4-cell stage. SN- and NSN-like oocytes have also been described in the antral compartment of human, rat, monkey, pig, and bovine ovaries. The findings reported in this paper may contribute to improved procedures for in vitro fertilization of humans and farm animals.     

The effects of alcohol on the heart: detrimental or beneficial?

Alcohol abuse is a substantial public health problem, resulting in staggering financial costs and other burdens. Prolonged alcohol abuse leads to cardiomyopathy in a minority of alcohol abusers, but because alcoholism is so widespread, alcohol is the major cause of nonischemic cardiomyopathy in Western society. In contrast, substantial evidence now suggests that moderate alcohol consumption has cardioprotective effects. It not only reduces the incidence of fatal ischemic heart disease, but it improves outcome in patients who have other risks for coronary events and go on to have myocardial infarctions. Therefore, physicians' recommendations about alcohol consumption should be as individualized as their patients.     

Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.     

Histochemical investigations on lectin binding in normal and irradiated mouse embryos

Human cancers express organ-specific neoantigens (OSNs) which elicit specific cellular immune responses in the cancer patient, as demonstrated by leukocyte adherence inhibition (LAI), an in vitro immune response assay. A purified protein of MW 40,000 (p40) exhibiting OSN (colon specific) activity was cleaved into specific peptide fragments and their partial amino acid sequences determined. This information was used in the polymerase chain reaction (PCR) to obtain a 992 bp cDNA clone (PCR-992) from a human colon adenocarcinoma cell line (LS-180). By comparison of the predicted amino acid sequence of PCR-992 with the known sequence of p40 peptides, PCR-992 was shown to correspond to almost the entire coding region of p40. Nucleotide sequence analysis suggested that the protein was mycoplasmal in origin due to its high A+T content (76%) and the presence of five in frame TGA termination codons; at least two of the latter are actually read as tryptophan, a known feature of mycoplasma translation. We have confirmed this origin by direct isolation of a contaminating mycoplasma species from the LS-180 cell line and demonstration that it could be hybridized with the PCR-992 probe. Northern and PCR analysis of RNA preparations from the contaminated LS-180 cell line showed that p40 was part of the high affinity transport system operon of Mycoplasma hyorhinis (Dudler et al, EMBO J., 7: 3963-3970, 1988). Total protein lysates of Mycoplasma hyorhinis cultivated without animal cells could elicit positive LAI responses when incubated with cancer patient leukocytes but not with normal patient leukocytes. The organ-specific nature of the response was, however, not observed indicating that host cell-mycoplasmal interactions may play a role in determining the organ-specific nature of p40 seen with the LAI. The significance of these findings will be discussed in the context of previous thinking regarding the origin of OSNs.     

The lack of long-term detrimental effects on liver allografts caused by donor-specific anti-HLA antibodies

Recent reports indicate a higher incidence of both acute and chronic liver allograft rejection when, at the time of transplantation, the recipients serum contains donor-specific anti-HLA antibodies. From 9/89 to 5/91, 133 liver allografts were performed at our institution. Thirteen liver recipients had donor-specific IgG anti-HLA antibodies (complement-fixing) at the time of transplantation. In eleven patients, antibodies reacted to donor class I antigens while in 1 patient the donor-specific antibody had class II reactivity. Twelve patients have been followed for a minimum of 12 months (median 18 months, range 28-12 months). No hyperacute rejection was seen in any of the cases and four patients had acute rejections. Thus far only one of the twelve patients has biopsy evidence suggestive of chronic liver injury. The remaining have normal liver enzymes and bilirubin. Three of these twelve patients died (one from a myocardial infarction and the others from sepsis) accounting for a one-year graft survival of 75%. There was no significant statistical difference in the one-year graft survival in those recipients without donor-specific antibodies (i.e., 80.5%). In eight of the twelve patients, pretransplant preformed antibody level (PRA) was > 50%. In six of the thirteen patients donor-specific antibody was present at dilutions greater than 1:64. As previously reported, the donor-specific antibody disappeared from the serum posttransplant within hours and did not reappear. In vitro studies demonstrated no factor in portal or hepatic artery blood that could inhibit rabbit complement mediated lysis of anti-HLA antibodies. We conclude that it is not a contraindication to do liver transplants in the presence of donor-specific anti-HLA antibodies.     

Mechanism of detrimental effects of carbon content on cleavage fracture toughness of low-alloy steel

The variation in fracture toughness of low-alloy base steels and weld steels with carbon contents of 0.08 and 0.21 wt pct was investigated using notched and precracked specimens tested at low temperatures. The attention is focused on the mechanism associated with detrimental effects on cleavage fracture toughness resulting from increasing carbon content. Analyses reveal that, in the case of constant ferrite grain sizes with increasing carbon content, the yield stress σ _y increases and the local fracture stress σ _f remains constant for notched specimens. For precracked specimens, the σ _y increases, whereas the σ _f decreases. In both cases, the ratio σ _f /σ _y decreases; this ratio is one of the principal factors inducing the deterioration in the cleavage fracture toughness of the higher carbon steels. Analyses also reveal that the critical strain for initiating a crack nucleus, which decreases with increasing carbon content and impurity elements, appears to be another principal factor that has a negative effect on the fracture toughness in both notched and precracked specimens. The results of the fracture toughness measured for weld metal with various grain sizes further support the predominant effect of grain size on the toughness of notched specimens.     

The in-vitro effects of nicotine and cotinine on sperm motility

Swim-up spermatozoa from the seminal samples of non-smokers, usually not exposed to passive smoking, were treated in vitro with nicotine (NIC) and cotinine (COT) at the average levels found in smokers' seminal plasma and at levels 500 times higher than this average. This was done to evaluate the action of these drugs on sperm motility. Each sample was allowed to swim up in Tyrode's solution with or without the drug; the study was carried out at time 0 and +1, +2, +4, +8 and +24 h of incubation, using a light microscope and a CASA system (experiment 1). In addition, the direct action of smoke on spermatozoa was studied using aspirated cigarette smoke (experiment 2). Kinetic parameters were then measured at 30 min, 45 min and 60 min starting from the last smoke injection. The first experiment showed that NIC and COT at average levels did not produce statistically significant variations of the kinetic parameters studied up to 24 h. However, the much higher concentration significantly altered all the kinetic variables in relation to the time of incubation. The second experiment with smoke in toto demonstrated a sharp reduction in all the sperm kinetic parameters. This reduction was seen after 30 min exposure to smoke and increased progressively until almost complete immotility at 1 h of exposure. These results suggest that NIC and COT are not responsible for the harmful effects of cigarette smoke on sperm kinetic parameters reported in the literature.