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.

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.

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.

Onconase (ONC) and bovine seminal ribonuclease (BS-RNase) are homologs of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, ONC and BS-RNase can evade the cytosolic ribonuclease inhibitor protein and are potent cytotoxins. Here, the endogenous cytotoxic activities of ONC and BS-RNase are compared in a wide variety of assays. Injections of ONC into one or both testes of mice and rats evokes a stronger aspermatogenic activity than does the injection of BS-RNase. Epididymides exposed to ONC lose mass and all sperm. Testicular tissue is gradually colonized by immunite and fibrocytic cells. Yet, Leydig cells are always present and functional in the ligamented parts of testicles injected with ONC or BS-RNase. ONC is likewise more toxic to mouse embryos than is BS-RNase, both in vitro and in vivo. The antiproliferative effect of ONC on human tumor cell line ML-2 and lymphocytes in a mixed lymphocyte culture is also more pronounced than is that of BS-RNase. The number of granulocyte-macrophage colony-forming units is repressed almost completely by ONC, whereas a five-fold higher dose of BS-RNase does not cause substantial inhibition. In mice, ONC is less immunogenic than BS-RNase but more immunogenic than RNase A. Together, these data indicate that ONC is a pluripotent cytotoxin, and serves as the benchmark with which to gauge the cytotoxicity of other ribonucleases.

Lamb ovaries represent an alternative source of sheep oocytes. The value of this source, however, is lowered by frequent incidence of polyspermy and other fertilization defects in embryos originating from these oocytes. The aim of this study was to determine if the pre-incubation of oocytes with different protein supplements (sheep serum, fetal calf serum, bovine albumin, heat-inactivated oviductal fluid, lyophilized oviductal proteins) will effect fertilization results in vitro. The total penetration rate was not affected by these treatments, while lyophilized oviductal proteins and bovine serum albumin increased monospermic fertilization to approx. 60%. In fetal calf serum this proportion was significantly lower (26%). Our results indicate that one of the most important components of the culture system affecting fertilization competence of juvenile lamb oocytes is protein supplementation, where sera are the most questionable part of the culture media. However, effective alternatives (bovine albumin or lyophilized proteins of oviductal fluid) could be used.

The aim of this study was to test the viability of embryos of two pronuclei stages after transfer to recipients. Experiments were carried out on 300 oocytes recovered from ovaries of Polish Longwool sheep. 76.6% (230) of the oocytes underwent the maturation process successfully. The cytological evaluation of the embryos revealed a high rate of fertilisation. In 80% (184/230) of the embryos both pronuclei and the tail in the vicinity of the male pronucleus were present. The poly sperms exceeded 10%. To test developmental ability, 72 embryos after two pronuclei stages were transferred to 12 recipients; 3 embryos were introduced into each oviduct of the recipients. As a result, 9 (75%) pregnant ewes delivered in term and 14 lambs were born. As a control, 20 Polish Longwool sheep were synchronically naturally inseminated, which resulted in 18 sheep delivering 29 lambs. These results demonstrate that sheep embryos can be produced in vitro, and after being cultured to only two pronuclei stages they can be successfully transferred to recipients.

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.

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 provides natural environment for gamete transport, fertilization and early embryonic development. The information about composition of milieu in the oviduct at defined stage of estrus cycle is valuable for the improvement of media employed in mammalian embryo production in vitro and for the investigation of sperm-egg interaction at fertilization. The intention of these studies was to collect oviductal secretion for biological assays and for chemical analyses using permanent indwelling oviductal cannulae in 14 merino ewes (28 oviducts). In 4 females (8 oviducts) with previously synchronized estrus cycle the harvest of secretion was low (maximal average 0.3 ml/day) and the flow stopped before day 7 after surgical intervention, mostly due to the oclussion by fibrinous plugs. The effect of unilateral ligation localized close to the uterotubal junction was compared in 6 ewes (6 ligated and 6 unligated oviducts). The ligation of the oviducts promoted the function of the canulae for several subsequent estrous cycles, while in unligated counterparts the cannulae became usually occluded with fibrin mostly before third cycle. In remaining 4 females (8 oviducts) in random stage of estrous cycles bilateral ligation was performed. The results show that this treatment was most suitable for collection of oviductal fluid. Despite fluctuation, in 8 from totally ligated 14 oviducts the undisturbed function of cannulae was recorded for more than 2 estrous cycles and in two instances for 150 days. The harvested volumes of secretion were in close relation to the estrous cycle with typical highest amount around the heat (1.2 mi per day) and lowest at the luteal phase (0.3 ml/day). The causes of flow cesation were mostly the formation of fibrinous plugs at the junction with oviduct or evulsion of cannulae by the subject. Nevertheless, the experiments indicate that cannulae may represent effective method for collection of oviductal secretion for biochemical analysis or direct physiological experiments.

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.

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.

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. Mol. Reprod. Dev. 48:344–349, 1997. © 1997 Wiley‐Liss, Inc.

The effect of exogenous bovine recombinant somatotropin (bST) on the recruitment of follicles and quality of oocytes was investigated in the bovine. Single injections of recombinant depot form of bST were given to nine heifers and seven cows at the time of synchronized estrus (day 0). Nine heifers and six cows served as controls. Three days after bST injection the animals were slaughtered, their ovaries dissected and follicles separated into two categories according to diameter (2–5 and 5–10 mm). A total of 196 (53.2%) healthy oocyte-cumulus complexes (OCC) was isolated and selected from 369 follicles (diameter 2–5 mm) of bST treated heifers and 137 (49.8%) OCC from 275 follicles of bST treated cows. After 25–28 h of culture, 164 (44.4%) and 124 (45.1%) oocytes from follicles of heifers and cows, respectively, had developed to the metaphase of the second meiotic division (MII). From control heifers, 122 (35.5%) OCC derived from 344 follicles (2–5 mm) and from control cows 87 (39.7%) OCC derived from 219 follicles were similarly selected for maturation in vitro and 109 (31.7%) and 78 (35.6%) oocytes in control heifers and cows, respectively, developed to the MII stage. The numbers of follicles developed in the experimental and control groups were similar (P > 0.05) but differences were evident in the healthy OCC and oocytes maturing in the treated versus control groups of heifers and cows (χ2 detected at 1% and 5% levels, respectively). The injection of bST resulted in significantly increased levels of growth hormone (GH) in peripheral blood and follicular fluid (FF), the GH levels in blood plasma correlating with the levels in FF. The level of insulin-like growth factor I (IGF-I) in sera from bST treated animals was significantly higher than in sera from control animals (353.6 vs. 155.5 ng ml−1). In conclusion, recombinant bST positively affected (at day 3) the proportion of OCC and oocytes maturing to MII.

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.

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.

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.

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 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.