Development of one-cell fertilized sheep ova following microinjection into pronuclei

Three experiments were conducted to identify, sources of loss of fertilized single-cell sheep eggs microinjected with DNA. In the first experiment, immediate transfer of eggs into synchronous recipients resulted in 86% of embryos developing (>32 cells) at Day 7. Incubating eggs in microdrops of Ham's F-10 medium + 10% fetal calf serum for 5 h at 37 degrees C in an atmosphere of 95% air: 5% CO(2) before transfer reduced development (65% >32 cells). Removing eggs from drops for 30 min of microscopic inspection, simulating manipulation during microinjection, caused no additional reduction in development (63% >32 cells). However, injection of eggs with buffer was detrimental to subsequent development (42% >32 cells). In Experiment 2, injection of buffer or injection of DNA in buffer into the pronuclei before transfer of eggs into recipient ewes resulted in 29 and 19%, respectively, of embryos developing to >32 cells at Day 7. In Experiment 3, more eggs developed when held in 5 ml of medium than in microdrops (P = 0.07). No difference in development was found between eggs held in bicarbonate-buffered BMOC or in phosphate-buffered saline with added fetal bovine serum. The development of sheep eggs appears to be greatly reduced after microinjection, but until alternate procedures are found, a high rate of loss of injected eggs may be an unavoidable cost of inserting foreign genes into sheep.     

Transplantation of nuclei into mammalian ova

A review of experimental studies of nuclear transplantation in mammals. The possibility of parthenogenesis in mammals is discussed on the basis of the results reviewed. Discrepancies in the data obtained by different authors are also discussed.     

The ability of dehydrated hamster and human sperm nuclei to develop into pronuclei.

To determine whether the nuclei of mature mammalian spermatozoa are resistant to dehydrated conditions, nuclei of hamster and human spermatozoa were freeze-dried or treated with various dehydrating agents before injection into hamster oocytes. Freeze-dried nuclei remained capable of developing into pronuclei even after 12 mo of storage at 4 degrees C. The level of DNA synthetic activity in the sperm (male) pronucleus was comparable to that in the egg (female) pronucleus. Sperm nuclei that had been stored in 100% ethanol, 100% methanol, or chloroform-methanol (2:1) mixture for 20 days were also capable of developing into pronuclei. Even the nuclei that had been dehydrated ("fixed") with Carnoy's fluid could develop into morphologically normal pronuclei. However, the level of DNA synthesis in the pronuclei derived from these chemically dehydrated nuclei was generally lower than that in the female pronuclei. Although the genetic integrity of the dehydrated sperm nuclei is yet to be determined, nuclei of mature hamster and human spermatozoa appear to be fairly resistant to dehydrated conditions.     

Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.     

Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.     

Predominance of beta-structure in solubilized zona pellucida from porcine ova

The isolated zona pellucida from porcine ova was effectively solubilized in water at 60 degrees C within one hour. The circular dichroic spectra of zona in water with and without dithiothreitol showed the beta-form. Although sodium dodecyl sulfate partially induced helical structure, the beta-form was considerably retained. These results indicate that the zona glycoproteins have a structure-forming potential for the beta-structure and the hydrogen bonds of the beta-structure stabilize the supramolecular complex of the zona pellucida. The beta-form was also detected in zona solubilized by tryptic digestion. Porcine acrosin, however, did not solubilize the zona.     

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 microL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst.     

Development of centrifuged cow zygotes cultured in rabbit oviducts

Zygotes from superovulated cows were centrifuged and pronuclei were detected by differential interference-contrast microscopy in 73% of 106 zygotes. Zygotes were then transferred to ligated oviducts of follicular-phase, 1-day pseudopregnant or 7-day pseudopregnant rabbits and recovered 5 days later. Their development did not differ from that of uncentrifuged zygotes transferred to the opposite oviduct: 41% of the embryos recovered from rabbit oviducts contained 17-32 nuclei and an additional 5% contained greater than 32 nuclei. In another experiment, 399 ova from unmated cows were transferred to rabbit oviducts to determine whether centrifugation induced parthenogenetic development. After 7 days, 257 ova were recovered; 16% of the recovered ova had developed parthenogenetically and contained 2-30 nuclei. Neither centrifugation of the ova nor reproductive status of the rabbits influenced the proportion of parthenogenotes found. Parthenogenetic development was also observed in 14 of 71 ova (20%) recovered on Day 7 from uninseminated superovulated cows. In an attempt to increase the probability of detecting treatment differences, centrifuged and control cow zygotes were incubated for 7 (rather than 5) days in opposite oviducts of fourteen 1-day pseudopregnant rabbits. Development was unaffected by centrifugation: 61% of the zygotes recovered had developed beyond the 16-cell stage, with 23, 24 and 15% containing 17-32, 33-64, and greater than 64 nuclei, respectively. Taking into account the percentage of zygotes in which pronuclei can be seen, the recovery rate from rabbit oviducts, and the proportion of embryos that develop to the morula stage or beyond, 26% of the original group of zygotes would be candidates for transfer into recipient cows.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

On the centering of the nuclei in centrifuged eggs as a result of fertilization and artificial membrane formation

Summary Migration of the egg-nucleus and of the fusion-nucleus was studied in the centrifuged eggs of two echinoids,Temnopleurus andDendraster. The evidence shows that the nucleus is pulled rather than pushed, and that the means of movement are probably contractile structures of the cytoplasm. In eggs which had been drawn into flask shape by centrifuging, if the sperm entered at the pole opposite that containing the egg-nucleus, the two nuclei did not meet, and subsequently only the part containing the sperm-nucleus showed segmentation. Eggs which had been caused to form membranes by chemical means required approximately twice the time of the normal for centering of the nucleus.     

Effect of pronuclear DNA microinjection on the development of porcine ova in utero

The objective of this study was to assess the effect of various aspects of pronuclear DNA microinjection on the early development of porcine ova in utero. Estrus was synchronized and superovulation was achieved in sexually mature gilts by the administration of allyl trenbolone, PMSG and hCG. Donor gilts were bred at 12 and 24 h after the onset of estrus. Ova were recovered between 60 and 62 h after the administration of hCG. One-cell ova that exhibited pronuclei after centrifugation were randomly allocated in equal numbers from each donor across one of two pairs of treatments: micro-DNA (ova were injected with two gene constructs that code for the human complement regulatory proteins decay accelerating factor and membrane cofactor protein) and control (ova were centrifuged only) or micro-buffer (ova were injected with buffer only) and pierced (a pipette was inserted into one pronucleus). Ova were transferred by treatment pairs to recipients. Treatments were segregated by oviduct. Ova were recovered after 120 h in utero, fixed and stained with 1% orcein. The proportion of ova that possessed > or = 80 nuclei, the mean number of nuclei present and proportion of ova that formed blastocysts were all significantly (P<0.05) greater for control and pierced ova than for micro-DNA and micro-buffer ova. No difference in these parameters was observed between micro-DNA and micro-buffer ova. These results demonstrate that pronuclear microinjection of a buffer alone can adversely affect the early development of porcine ova in utero.     

Development of pronuclei in pig oocytes activated by a single electric pulse.

Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.