Biomedical subjects

V Landa

Publications and source records attributed to V Landa.

At least 37 records · Page 2Linked to original sources

PubMed1991

Immunosuppressive factor in cow follicular fluid.

The activity of the immunosuppressive fraction and proteinase inhibitor isolated from cow follicular fluid was investigated. The immunosuppressive factor was separated from the accompanying proteinase inhibitor by Sepharose 6B and Sephacryl S-200 column chromatography. In vitro, the fraction significantly reduced mitogen-induced lymphocyte proliferation. In vivo, the fraction inhibited mouse plaque formation. However, the factor had no effect on the development of intact or zona free embryos. The active components have a molecular mass of about 110,000.

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PubMed1991

The construction of different types of aggregates and chimaeric aggregates from individual blastomeres of mouse 8-cell embryos.

Different types of aggregates and chimaeric aggregates were constructed from individual 1/8-blastomeres of C57BL/6J and 129/Sv 8-cell embryos. Addition of phytohaemagglutinin into manipulation medium allowed us to construct large numbers of 4/8 to 9/8 aggregates and chimaeric aggregates exactly according to the design of the experiment. The developmental potential of reconstructed embryos was high. Over 93% of 4/8 to 8/8 aggregates and 90% of different types of 7/8 to 9/8 chimaeric aggregates formed blastocysts during 30 to 34 h of in vitro culture. All types of 4/8 to 8/8 aggregates and 7/8 to 9/8 chimaeric aggregates, in vitro cultured for 24 h, were able to undergo normal implantation after transfer into day-3 recipients. Live young were born after the transfer of morulae and blastocysts developed from 8/8 aggregates, and chimaeric morulae and blastocysts formed from different types of 7/8 to 9/8 chimaeric aggregates. All young born after the transfer of chimaeric embryos were overt C57BL/6J in equilibrium 129/Sv chimaeras.

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PubMed1991

Cryopreservation of mouse zona-free embryos and embryos exposed to biopsy and reconstruction by vitrification in microdrops.

Zona-free zygotes, 2-cell, 4-cell, and 8-cell embryos, morulae, blastocysts and embryos exposed to biopsy and reconstruction were cryopreserved in microdrops of vitrification medium expelled directly into liquid nitrogen. While none of the zygotes and 2-cell embryos survived storage in liquid nitrogen, 54% 4-cell and 97% 8-cell embryos, 99% morulae, 88% blastocysts, 98% biopsied and 100% reconstructed embryos were intact one hour after thawing. Developmental potential of cryopreserved zona-free embryos evaluated after 30 h of in vitro culture was high. Of the frozen embryos, 49% 4-cell and 92% 8-cell embryos, 95% morulae, 79% blastocysts, 84% biopsied and 90% reconstructed embryos were capable of further cleavage. After the transfer of zona-free 8-cell embryos, morulae, blastocysts, biopsied and reconstructed embryos into day-1 recipients - 4 (16%), 14 (56%), 3 (12%), 0 and 8 (32%) live foetuses were recorded, respectively.

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PubMed1991

Vitrification of intact and half 8-cell mouse embryos injected with DNA at 2-cell stage.

Two-cell embryos were collected from (C57BL/6 x CBA)F1 mice, microinjected with foreign DNA and transferred into temporary recipients. Intact and half 8-cell embryos recovered from oviducts 24 h later were frozen in microdrops of vitrification medium and stored in liquid nitrogen for 2 to 30 days. Development of frozen/thawed embryos was assessed by in vitro culture or transfer into day-1 recipients. From 1200 2-cell embryos 77% intact and 20% half embryos were obtained one hour after DNA injections. After transfer of 600 intact and 300 half embryos into temporary recipients, 77% intact and 73% half 8-cell embryos were recorded, respectively. Survival and in vitro development of frozen/thawed DNA-injected embryos were high, as 97% intact and 91% half 8-cell embryos developed to morulae or blastocysts. After transfer of 300 intact and 150 half 8-cell embryos into day-1 recipients, 99 (33%) and 19 (13%) young were born. None of the mice born from DNA-injected and cryopreserved embryos integrated injected genes.

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PubMed1990

[Evaluation of the degree of compliance in arterial hypertension protocol].

We report an evaluation of the hypertension protocol. Its aims were to assess the degree of compliance with its variables, the number, age and sex of hypertensives, and the rate in whom blood pressure levels were controlled in the patients included and not included in the protocol. The study was carried out in the Basauri Teaching Unit from January 1987 to June 1989. We reviewed all the parameters of the medical and nursing protocol of 1,429 patients diagnosed as hypertensive and the mean value of the three last blood pressure readings. A total of 67.2% of patients were included in the protocol. The compliance was higher than 80% in most of its items; 61.8% of the prescribed nursing controls were carried out. Blood pressure was less than or equal to 159/94 in 68.1% of patients. This rate was higher in the patients included in the protocol. The protocol was useful for the control of hypertension, although it should be modified directing it to primary care.

Adult↗

PubMed1990

Diploidization of bovine oocytes matured in vitro and parthenogenetically activated by electric shock.

Bovine in vitro matured oocytes were parthenogenetically activated by a single pulse of direct current. Suppression of second polar body extrusion by 60 V DC-pulse followed by cytochalasin B treatment or 240 V DC-pulse were used to diploidize parthenogenones. Analysis of the cytological events 20 h postactivation clearly documented high efficiency of both diploidization techniques. All oocytes exposed to a single 60 V DC-pulse and then to cytochalasin B for 3 to 6 h were activated and 70% to 88% possessed two pronuclei. Exposure to 240-V DC-pulse for 10 and 20 microseconds activated all oocytes, 72% and 62% formed two pronuclei, but a high incidence (22% and 30%) of degenerated oocytes was observed. Prolonged in vitro culture of oocytes after parthenogenetic activation and diploidization treatment showed that formed pronuclei were able to fuse and single prometaphase to telophase mitotic cleavage figures developed in all oocytes fixed 28 h after activation, except one cytochalasin B-treated oocyte with two prometaphase sets of chromosomes.

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PubMed1990

Cryopreservation of mouse 8-cell embryos in microdrops.

Eight-cell embryos collected from superovulated inbred strains and F1 hybrid mice were frozen by the microdrop technique developed in our laboratory. The technique based on pre-equilibration in medium with 10% glycerol, before transfer into vitrification solution, expel of embryos in 5 microliters to 20 microliters of vitrification solution directly into liquid nitrogen and thawing of microdrops in medium with 0.5 M sucrose was used. The behavior and morphological appearance of embryos during pre-freezing and post-thawing periods was documented. The efficiency of cryopreservation in microdrops was high, as documented by 90% to 100% of intact embryos after the freezing and thawing cycle. Furthermore, no zona pellucida damage was observed. The developmental potential of embryos frozen in microdrops was comparable with development of unfrozen embryos of the same genetic origin. After freezing and storage 83% to 93% of embryos developed to blastocysts and 73% to 92% embryos underwent "implantation" after 48 h and 96 h of in vitro culture, respectively.

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PubMed1990

Construction of aggregation chimaeras from 8-cell mouse embryos stored for several years in liquid nitrogen.

Eight-cell embryos of 7 different inbred strains frozen and stored for 2 to 4 years in liquid nitrogen were used as the source of embryos and individual 1/8-blastomeres for construction of different types of aggregation chimaeras. Overall survival of stored embryos, evaluated by in vitro culture, ranged from 63% in A/By to 85% in C57BL/10By embryos. Chimaeric embryos prepared by aggregation of frozen/thawed zona-free 8-cell embryos continued in normal development through expanding blastocysts (77% to 93%) to in vitro implantations. Construction of directed chimaeras by total rebuilding of 8-cell embryos previously stored in liquid nitrogen was successful and resulted in normal development. Of the aggregates made by regular mixing of 1/8-blastomeres obtained from BALB/c and C57BL/6J embryos thawed from liquid nitrogen, 90% reached the blastocyst stage and 77% implanted after 48 h and 96 h of in vitro culture, respectively. Addition of 1/8 blastomeres from frozen/thawed BALB/c embryos to four 1/8-like blastomeres from nuclear transplantation experiments documented, as all of the 30 constructed aggregates formed expanding blastocysts within 48 h of in vitro culture, the possibility of using embryos from embryo banks for the rescue of products of genetic manipulations at the embryo level.

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PubMed1990

"Two-step" freezing of individual blastomeres from mouse eight-cell embryos.

Eight-cell embryos recovered from superovulated C57BL/6J mice were disaggregated into individual 1/8 blastomeres. For cryopreservation of blastomeres a simple "two-step" technique based on direct transfer of blastomeres into the final concentration of DMSO (0.5 to 1.5 M) and exposure of plastic straws with blastomeres to -25 degrees C for 10 min before immersion into liquid nitrogen was used. The samples stored for 2 to 60 days were thawed at different temperature (20 to 80 degrees C) and intact 1/8 blastomeres were transferred into DMSO-free medium. The survival of blastomeres in individual groups varied from 52% to 90% according to the combination of DMSO concentration, exposure time to DMSO prior to freezing and temperature of thawing bath. Intact frozen-thawed 1/8 blastomeres were able to undergo cleavage and aggregates constructed from 4, 6, 7, and 8 blastomeres developed throughout preimplantation period. Of the aggregates constructed 1 to 2 h after thawing, 78% to 93% reached the blastocyst stage within 54 h of in vitro culture. Possible use of frozen-thawed 1/8 blastomeres in nuclear transplantation experiments and genetic manipulation on mammalian embryos is discussed.

Animals↗

PubMed1990

Parthenogenetic activation of mouse tubal oocytes by a single pulse of direct current.

Tubal cumulus-free oocytes recovered from C57BL/6J and BALB/c mice 16 to 24 h after hCG injection were parthenogenetically activated by a single DC-pulse of different voltage. The efficiency of activation was assessed 2, 4 and 24 h after electric stimulation according to the proportion of oocytes with second polar bodies, presence of pronuclei and development of parthenogenetic 2-cell embryos. The best results, 99% and 100% of oocytes with second polar body (2 h) or with second polar body and one pronucleus (4 h), were recorded when oocytes of both strains were activated by a single 60 V DC-pulse (30 microseconds) 20 h after hCG injection. Aging of oocytes or higher voltage of DC-pulse (120 V and 240 V) resulted in both strains in an increase of abnormal oocytes and oocytes with two pronuclei. While a large proportion of activated C57BL/6J oocytes were totally fragmented within 24 h and only 23% reached 2-cell stage, 78% of the activated BALB/c oocytes developed to parthenogenetic 2-cell embryos. It was shown that pronuclear oocytes and parthenogenetic 2-cell embryos for preparation of different types of cytoplasts could be effectively prepared by electric stimulation of tubal oocytes.

Animals↗

PubMed1989

Transplantation of nuclei from 2- to 16-cell embryos into enucleated blastomeres of 2-cell mouse embryos.

Individual blastomeres of 2-, 4-, 8- and 16-cell embryos were fused with enucleated blastomeres of 2-cell embryos. The percentage of fused nuclear donor/recipient pairs was high (92 to 100%). Most of the reconstituted embryos cleaved twice within 24 h of in vitro culture and 83% to 95% developed to the morula or blastocyst stage. Increasing the number of blastomeres in reconstituted embryos by the construction of aggregates from blastomeres obtained 24 h after nuclear donor/recipient fusion was effective. All constructed aggregates reached the morula or blastocyst stage within 30 h of in vitro culture. The normal day-13 implantations were recorded after the transfer of reconstituted "half" embryos and aggregates when 8-cell blastomeres were used as nuclear donors.

Animals↗

PubMed1989

Parthenogenetic activation of cattle oocytes by a single pulse of direct current.

Bovine follicular oocytes cultured in vitro for 26-27 h were exposed to AC/DC cycle. Mature and immature oocytes were effectively parthenogenetically activated by a single DC-pulse of a different voltage. Application of AC/DC cycle with 60 V DC-pulse (30 microseconds) resulted in 96% of activated mature and 94% of activated immature oocytes. The high activation rate was also recorded when oocytes were treated with higher voltage. The exposure of oocytes to 120 V and 240 V pulses increased the number of degenerated oocytes (6% to 46%) and incidence of oocytes with two pronuclei. The resumption of meioses was observed in 92% of mature and 88% of immature oocytes 1 h after DC-pulse and pronuclei formation was recorded in 72% of mature and 40% of immature oocytes after 6 h of IVC. Two-cell stage parthenogenones were obtained after prolonged IVC (22%) or after transfer into mouse oviducts for 44 h (61%). Possible use of parthenogenetic pronuclear oocytes and 2-cell parthenogenones in pronuclear/nuclear microinjection and nuclear transplantation experiments is outlined.

Animals↗

PubMed1989

Cytoplasts from two-cell embryos for nuclear transplantation in the mouse.

Cytoplasts for nuclear transplantation were prepared by microsurgical enucleation of one or both blastomeres of C57BL/6J and 129/Sv-ter 2-cell embryos. The survival of cytoplasts was high (85 to 97%) in both inbred strains. When cytoplasts with a "doubled" amount of cytoplasm were prepared by electrofusion of enucleated blastomeres in individual embryos, the fusion rate in C57BL/6J embryos was 93% while in 129/Sv-ter only 2%. Similar fusion rates were obtained for C57BL/6J (100%) and 129/Sv-ter (7%) embryos when enucleated and intact blastomeres were fused. The live foetuses and young born after transfer of manipulated embryos into day-1 recipients showed that enucleation and electrofusion were compatible with normal pre- and post-natal development.

Animals↗

PubMed1985

Two-step freezing of cells used in hybridoma technology.

Hybridoma technology requires freezing of parental myeloma cells, continuous freezing and thawing of clones and stable hybridoma cells. A modification of the method of two-step freezing is described. The cryoprotective agent (5% dimethyl sulphoxide) is added to the cells at room temperature for 10 min. Cells are then transferred directly to the -25 degrees C bath, held at this temperature for 10 min, and stored directly in liquid nitrogen. Thawing is rapid in a water bath warmed to 60-80 degrees C. Hybridoma cells retain high viability and the production of specific monoclonal antibody after thawing.

Animals↗

PubMed1982

Rapid thawing of rabbit embryos after storage at -196 degrees C.

Rabbit morulae frozen to -25 degrees C and transferred from this temperature to -196 degrees C were thawed at different rates. Maximum survival of the embryos was obtained at thawing rate of 650 degrees C/min. Slower and more rapid thawing decreased the survival. At thawing rates of 25 degrees C/min and 1 550 degrees C/min all morulae died. The ability of morulae to develop after storage at -196 degrees C and thawing at 650 degrees C/min was assayed by culture in vitro and transfer into the recipients. Of 241 morulae obtained after thawing, 197 (81.7%) continued normal development. Of 100 unfrozen morulae, 97 (97%) developed to the blastocyst stage. Of 41 frozen and 20 unfrozen embryos transferred into the recipients, 28 (68.3%) and 19 (95%), respectively, implanted. After transfer of 37 blastocysts obtained by culture of morulae thawed at 650 degrees C/min, 5 recipients have born 21 (56.8%) live offspring.

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PubMed1982

A simplified method for freezing mouse blastocysts.

Mouse blastocysts frozen slowly (0.3-0.5 degrees C min-1) to -25 degrees C and transferred to -196 degrees C survive long-term storage to the extent comparable to unfrozen blastocysts. After thawing at 450 and 650 degrees C min-1, 91 and 93% blastocysts, respectively, continued normal development under in vitro conditions. The length of equilibration at -25 degrees C before transfer to -196 degrees C had no marked effect on survival of blastocysts. It has been shown that blastocysts stored at -196 degrees C may be transferred into recipients immediately after thawing. The prerequisite of a normal development was the transfer into pseudopregnant females on day 2 of pseudo-pregnancy; 60.3% foetuses were found 14-15 days after transfer. Transfers of thawed blastocysts into pseudopregnant females on day 3 or day 4 of pseudopregnancy failed (3.9 and 0% foetuses, respectively). After transfer of unfrozen blastocysts into pseudopregnant females on day 3 of pseudopregnancy 58.7% foetuses were found. Transfers of unfrozen blastocysts into pseudopregnant females on day 2 and day 4 of pseudopregnancy were less effective (6.4 and 27.3% foetuses, respectively).

Animals↗

PubMed1982

Protection of non-cellular investments of rabbit morulae stored at--196 degrees C.

The non-cellular investments were damaged in only 2.8% of rabbit morulae frozen/ thawed in plastic straws. Similar results (4.8%) were obtained with glass ampoules only when morulae were slowly frozen and slowly thawed in siliconized ampoules. Freezing/thawing by other methods and the use of untreated ampoules resulted in high incidence of embryos with damaged investments (41-61%). Morulae frozen/thawed by a method allowing an almost complete elimination of investment injury displayed high survival when slowly frozen and rapidly thawed (90.7%) while slow freezing and slow thawing decreased survival (74.5%). Morulae subjected to rapid freezing were for the first time successfully stored at--196 degrees C. After rapid thawing 59.6% morulae survived. The mechanism of damage to the non-cellular investments of rabbit embryos is discussed.

Animals↗

PubMed1981

Factors influencing the results of transfers of rabbit embryos stored at -196 degrees C.

Rabbit embryos at the 8-cell and morula stages were frozen and stored at -196 degrees C for 2-200 days. After thawing the embryos were examined for their viability in vitro and in vivo. In vitro, 62.5% of frozen 8-cell embryos and 81.4% of frozen morulae developed to blastocysts. In the control group of unfrozen embryos, 93.2% 8-cell embryos and 92.4% morulae developed to the blastocyst stage. Culture permitted a more reliable elimination of the embryos damaged during freezing and thawing. Embryos were transferred into the reproductive tracts of the recipients either directly after thawing or after 24 h in culture. Synchronous transfers of frozen rabbit embryos were not successful. After asynchronous transfers of morulae and blastocysts into the oviducts, implantation was 31.8% and 42.9%, respectively. After transfer of blastocysts into the uterine horns of the recipients, 47.6% embryos implanted.

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