Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

An attempt to reduce polyspermic penetration in lamb oocytes.

The incidence of polyspermy in lamb oocytes matured and fertilized in vitro is very high and this results in a reduced developmental potential of embryos arising from them. We have attempted to produce oocytes more resistant to this fertilization anomaly. The oocytes from prepubertal lambs 7-12 weeks old were matured in a medium supplemented with various blood sera and oviductal fluid and fertilized in vitro. Significantly higher monospermic penetration was found in a medium supplemented with BSA--3 mg/ml (63.9%) and OF--20% concentration (55.8%). Lower monospermy was recorded in the presence of 10% LS (44.6%) or 10% SS (40.8%), and particularly in a medium with 10% FCS (26.9%). In contrast, high monospermy (78.7%) was observed in oocytes from adult donors matured and fertilized in an identical system. In another set of experiments we estimated whether polyspermy can be reduced by improvement of the cytoplasmic maturation of prepubertal oocytes using a two-step maturation protocol. After artificial arrest of the maturation for 24 h with a specific cdk inhibitor--BL-I, 50 miocroM--more than 80% oocytes from prepubertal and adult donors did not resume meiosis. When incubated thereafter in a drug-free medium for another 24 h, the oocytes of both categories progressed to MII in the rate comparable with control (80% to 90% MII). However, after fertilization no significant differences in the level of monospermic penetration was recorded between the arrested group (59.8%) and control (58.8%), both matured in the presence BSA, and 46.6% and 52.3% after treatment with OF. Also, no significant difference was observed between the arrested and control oocytes from adult donors (72.6% and 84.8%, respectively). These results suggest that high polyspermy in prepubertal oocytes is caused by developmental imperfection and can't be fully eliminated either by modifying the composition of culture media or by prolongation of the culture interval.

Influence of oviductal fluid on ovine embryo viability.

The severe loss of developmental competence affecting fertilized ova when removed from the oviductal environment suggests that this organ plays a functional role in early embryonic development. The purpose of this study was to determine the effect of sheep heat-inactivated OF on the mortality rate of ovine embryos produced in vitro and transferred into recipients. As control groups we used embryos fertilized and cultured in media supplemented with different kinds of proteins (FCS, BSA). Transfer of embryos in the two pronuclei stage to the oviducts of synchronized recipients resulted in 60% of successfully termed pregnancies after incubation of embryos in OF, 40% in BSA and only 10% after FCS. All ewes were further assessed for pregnancy by ultrasonography 33, 53 and 80 days after embryo transfer. The highest embryo mortality appeared between day 33 and 52. We concluded that incubation of ovine oocytes in OF during the final period of the maturation process may play a functional role at the time of fertilization and early embryonic development.

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation.

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.

Bovine seminal ribonuclease attached to nanoparticles made of polylactic acid kills leukemia and lymphoma cell lines in vitro.

Bovine seminal ribonuclease (BS-RNase) is a protein with a number of biological effects. It shows antitumoral, aspermatogenic, antiembryonic, immunosuppressive and antiviral properties. The cytotoxic effects appear to be specific for tumor cells as non-malignant cells seem to be unaffected in vitro. Unfortunately, the in vivo application of BS-RNase so far was successful only when it was administered intratumorally. Therefore, the objective of the present investigation was to improve the properties of BS-RNase by attachment to nanoparticles made of polylactic acid (PLA-NP) using an adsorption method. This preparation was tested in vitro against leukemia (MOLT-4) and lymphoma (H9) cell lines sensitive and resistant to cytarabine. No difference between the nanoparticle preparation and pure BS-RNase was found in these tests. To examine the in vivo effects, the preparations were tested for their aspermatogenic and antiembryonal efficacy compared to the pure BS-RNase as a rapid test for antitumoral activity. The aspermatogenic and antiembryonal effects were enhanced by the nanoparticle preparation. Consequently, BS-RNase loaded adsorptively to PLA-NP holds promise for the in vivo use as an antitumoral agent. Further research will investigate the efficacy of this preparations in an in vivo tumor model.

Oviduct secretion contributes to the establishment of species specific barrier preventing penetration of oocytes with foreign spermatozoa.

Ovulated oocytes can be fertilized in vivo almost exclusively with spermatozoa of its own species only, while the species specificity of zona pellucida of in vitro matured oocytes is less restrictive. Our present experiments were undertaken to determine whether estrous oviductal fluid modifies the interaction between gametes of unrelated species. After incubation of in vitro matured sheep oocytes with bull spermatozoa, the penetration rate was 75.0%, whereas when the oocytes were matured in medium supplemented with 15% sheep oviductal fluid collected using the permanent indwelling oviductal cannulae, the penetration rate decreased to 4.8 % (4/84). In reverse combination, 70.4 % (38/54) cattle oocytes matured in vitro were penetrated with ram spermatozoa. The addition of oviductal fluid caused a drop in penetration by ram sperm to 38% (19/50). In parallel experiments, no penetration was recorded when in vivo matured sheep oocytes were incubated with bull spermatozoa; high fertilization rates (79.4% - 27/34) were recorded when such eggs were incubated with ram spermatozoa, irrespective to the presence or to the absence of oviductal fluid in the medium. The results suggest that the properties of zonae pellucidae of ovulated and in vitro matured oocytes are not identical and may be modified by contact with estrous oviductal fluid.

Solitary fibrous tumor of the meninges occurring after irradiation of a mixed germ cell tumor of the pineal gland.

Twenty-nine months after surgery, irradiation, and systemic chemotherapy for a pineal mixed germ cell tumor, an 11-year-old Caucasian male developed a 3 cm dural based nodule in the occipital lobe that proved to be a solitary fibrous tumor by immunohistochemical and ultrastructural examination. Differential diagnosis included fibrous meningioma, neurofibroma, Schwannoma, cranial fasciitis of infancy, and solitary fibrous tumor. A Masson trichrome stain revealed a prominent collagenous stroma and reticulin staining exhibited strong pericellular positivity. Immunohistochemical staining demonstrated diffuse vimentin and focal CD34 positivity of tumor cells. Ultrastructural examination revealed fibroblastic differentiation. These features are consistent with solitary fibrous tumor. Although we favor a radiation-induced origin for the neoplasm, alternative explanations for the tumor's origin include cerebrospinal fluid spread from the original germ cell tumor or a de novo neoplasm.

Developmental failure of hybrid embryos originated after fertilization of bovine oocytes with ram spermatozoa.

The developmental ability of hybrid zygotes, produced by in vitro fertilization of in vitro matured bovine oocytes with ram sperm, was evaluated by gross morphology, autoradiographic detection of (5-3H) uridine incorporation, and fine structure morphology. Fertilization was successful in 83% of bovine oocytes inseminated with bull sperm (control embryos) compared with 67% of bovine oocytes inseminated with ram sperm (hybrid embryos) and in both cases appeared two regularly developed pronuclei. Two-cell embryos were transferred to ewe oviducts and allowed to develop to the 8-cell stage. Although the ability of hybrid embryos to reach 8-cell stage was similar to that of control embryos, in nuclei of hybrid embryos the transition from maternal to embryonic genome control assessed according to the onset of RNA synthesis indicated the differences in the frequency of labelled nuclei and intensity of their labelling. In hybrid embryos these parameters were remarkably lower and may reflect the developmental failure of hybrid embryos. These observations are consistent with delay or inefficient reactivation of the embryonic genome in the hybrid embryos.

Retrospective determination of HIV-1 status by a PCR method on paraffin wax embedded sections.

AIM: To develop a simple but reliable polymerase chain reaction (PCR) method to determine the HIV-1 status of patients on formalin fixed, paraffin wax embedded lymph node tissue. METHODS: Fifty lymph node specimens, 20 from HIV-1 seropositive and 30 from HIV-1 seronegative patients, were analysed. Lymph nodes with a variety of disease conditions were included in the study. Tissue sections were treated with a DNA extraction buffer containing proteinase K and the crude cell lysate was used in PCR analysis. Nested primers were used to amplify HIV-1 DNA sequences coding for gag, pol and env proteins. PCR products were demonstrated by polyacrylamide gel electrophoresis. Results were then compared with HIV-1 serology of the patients from whom the tissue was obtained. RESULTS: The PCR method yielded a specificity of 100%, a sensitivity of 95%, a positive predictive value of 100%, and a negative predictive value of 97% when compared with HIV-1 serology. The kappa statistic (0.958) showed an excellent agreement between the PCR method and serology. Furthermore, HIV-1 DNA was demonstrated in lymph node tissue from a serologically unconfirmed acquired immunodeficiency syndrome case necropsied in 1982. CONCLUSION: This PCR method is a simple and reliable means of retrospectively determining the HIV-1 status of patients using formalin fixed, paraffin wax embedded lymph node tissue.

In vitro fertilization of intact sheep and cattle oocytes with goat spermatozoa.

In an earlier study we reported on the fertilization of in vitro-matured bovine oocytes by ram spermatozoa. The present results extend those observations and demonstrate the penetration of bovine and ovine oocytes matured in culture by goat spermatozoa. Freshly ejaculated and in vitro capacitated goat spermatozoa penetrated 37.1% of bovine and 48.1% of ovine intact oocytes. Monospermic fertilization was detected in about 90% of the oocytes of both species, and the development of pronuclei followed a developmental pattern similar to that of homologous fertilization.

Pregnancy rate after the transfer of sheep embryos originated from randomly chosen oocytes matured and fertilized in vitro.

Randomly chosen sheep oocytes isolated from 2- to 5-mm follicles of hormonally nonstimulated slaughtered females were matured and fertilized in vitro. Using heparin for the induction of ram sperm capacitation, a fertilization rate close to 80% was recorded. After the transfer of 29 embryos cultured to the 2- to 4-cell stage to 4 recipients, each delivered 1 lamb. In another experiment, 34 2-cell embryos stage were transferred (1 to each oviduct) to 17 synchronized recipients; 8 pregnancies were established and each of 5 recipients delivered a single lamb. The remaining 3 recipients aborted at the third month of gestation. These results show that sheep embryos can be produced in vitro from randomly chosen oocytes and by using relatively simple procedures. However, the viability of the embryos was low, with approximately only 15% developing to term after transfer at the 2-cell stage.

Pregnancies after transfer of sheep embryos produced from oocytes matured and fertilized in vitro.

The experiments describe simple and effective methods used for the production of sheep embryos in vitro. The oocytes isolated from the ovarian follicles, 2-5 mm in diameter, were matured in culture for 22-24 h. After mixing with ram spermatozoa pretreated with heparin, 77-100% of fertilized oocytes were monospermic. Some of them (60-68.3%) cleaved in culture after 24-50 h to 2-4-cell stage. The transfer of cleaved embryos to the oviducts of 4 synchronized recipients resulted in pregnancies and 4 normal lambs were born at term.

Penetration of intact bovine ova with ram sperm in vitro.

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.

The use of transvaginal ultrasonography in the diagnosis of ectopic pregnancy.

Despite advances in diagnosis made by the introduction of serum beta-subunit of human chorionic gonadotropin determinations and transabdominal ultrasonography, ectopic gestations still present a major diagnostic challenge. The increased resolution of the transvaginally introduced high-frequency ultrasound transducer probes seems to solve this diagnostic problem. In this study 145 patients were referred for ultrasonographic workup because of a suspected ectopic gestation. In 38 patients a diagnosis could be made with classical transabdominal scanning. One hundred seventeen patients required additional transvaginal scanning with a 5.0 and a 6.5 MHz probe. In 98 patients a diagnosis was made during the first transvaginal scan; nine patients were rescanned within 3 days for the final diagnosis. In 56 patients, ectopic pregnancy was successfully ruled out by transvaginal scanning. Thirty-nine ectopic pregnancies were diagnosed. Only one false-positive identification was made. The sensitivity of diagnosing ectopic pregnancy by high-frequency transvaginal sonography was 100%; the specificity was 98.2%. The positive predictive value of this method was 98%, and the negative predictive value was 100%. The rate of the beating fetal heart was seen in the tube (23%). The high number of unruptured tubal pregnancies in this series (66%) suggests the possibility of an early diagnosis that may have therapeutic implications. The use of higher-frequency transvaginal transducer probes improves the diagnosis of the ectopic gestation.

Effect of glycerol on the penetrating ability of fresh ram spermatozoa with zona-free hamster eggs.

The presence of 10% glycerol in diluted semen for 30-90 min before preincubation stimulated sperm penetration of zona-free eggs. Prolongation of semen storage in medium with glycerol did not further improve penetration ability. The penetrating activity of spermatozoa depended on the concentration of glycerol and incubation time. These results indicate that the presence of glycerol accelerates induction of the acrosome reaction which could be one of the causes of lower conception rate after insemination with deep-frozen ram semen.