Development of enucleated mouse oocytes reconstituted with embryonic nuclei

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.     

Spermatozoan response to the toad egg matured after removal of germinal vesicle.

The germinal vesicle (GV) was removed from toad oocytes at various times after treatment with progesterone, and enucleated eggs were inseminated under conditions that ensured fertilization of nucleated control eggs. The eggs enucleated before the initiation of GV break-down did not show genuine cleavage. Cytological examinations revealed, however, that spermatozoa enter the eggs enucleated even before the hormone treatment, without subsequent formation of pronuclei or DNA synthesis. The same lack of response was observed when several detergent-pretreated sperm were injected into enucleated eggs. When GV material was injected back into enucleated oocytes, the injected spermatozoa underwent transformation and DNA synthesis, although in variable degrees, in the egg cytoplasm. It is concluded that the egg becomes fertilizable independently of the GV during the hormone-induced maturation process. After entering the egg, however, spermatozoa require GV material for their participation in the developmental process.     

Nuclear transplantation of male primordial germ cells in the mouse

We examined the developmental ability of enucleated eggs receiving embryonic nuclei and male primordial germ cells (PGCs) in the mouse. Reconstituted eggs developed into the blastocyst stage only when an earlier 2-cell nucleus was transplanted (36%) but very rarely if the donor nucleus was derived from a later 2-cell, 8-cell, or inner cell mass of a blastocyst (0-3%). 54-100%, 11-67%, 6-43% and 6-20% of enucleated eggs receiving male PGCs developed to 2-cell, 4-cell, 8-cell and blastocyst stage, respectively, in culture. The overall success rate when taking into account the total number of attempts at introducing germ cells was actually 0-6%. Live fetuses were not obtained after transfer of reconstituted eggs to recipients, although implantation sites were observed. The developmental ability of reconstituted eggs in relation to embryonic genome activation and genomic imprinting is discussed.     

Survival of maternal mRNA in anucleate and unfertilized mouse eggs

Unfertilized mouse eggs were parthenogenetically activated in vitro and then bisected. Anucleate fragments were aged in vitro, and their protein synthesis was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proteins were compared with those which were synthesized by aging unfertilized eggs and those which were translated in vitro from mRNA extracted from the unfertilized eggs. Normally cleaving parthenogenetic eggs served as controls. Cytoplasts and unfertilized eggs synthesized considerable quantities of protein after 2 days in culture. The protein patterns of cytoplasts and unfertilized eggs shifted in this time mainly within a group of proteins with a molecular mass of about 35 kDa. This shift was also seen in controls between day 1 and 2 but was delayed in unfertilized eggs. There was no clear appearance of new proteins in aging cytoplasts, which might have indicated a selective activation of maternal mRNA at a certain time after the activation stimulus, nor was such a change apparent in unfertilized eggs. The survival of maternal allozymes of glucose phosphate isomerase was tested in cytoplasts derived from fertilized eggs. The allozymes remained active during 4 days of aging and did not change their quantitative correlation.     

Insemination of immature sea urchin (Arbacia punctulata) eggs

Nuclei from osmotically opened erythrocytes and erythroblasts were injected into nucleated or enucleated Xenopus laevis eggs. Although the cleavage pattern of the recipient eggs which started to divide was normal in about half of the cases, nuclei from erythrocytes injected into nucleated or enucleated eggs never promoted development beyond the early gastrula stage. In contrast, nuclei from osmotically opened erythroblasts injected into enucleated eggs promoted development to early tadpole stages (stages 29–36). Frequently, injection of osmotically broken erythroblasts injected into nonenucleated eggs gave rise to triploid larvae which all died at roughly the same early tadpole stages (29–36). Surprisingly, development did not proceed to the stage of advanced organogenesis (stages 44–47), which is easily reached by gynogenetic haploids: The presence of the haploid genome derived from the egg pronucleus did not significantly improve the developmental capacity. Embryos obtained by single injection of erythrocyte nuclei into nucleated eggs were unable to pass the gastrula stage. To invalidate the interpretation that the observed arrest in development was related to nuclear damage during injection of the recipient eggs, single unbroken erythrocytes and unbroken erythroblasts were transferred into nucleated and enucleated eggs. No cleavage was observed in both classes of eggs injected with unbroken erythrocytes. In contrast, erythroblasts were found to induce cleavage in the recipient eggs at a frequency of about 11%. To ascertain that the nucleus of unbroken erythroblasts participated in development, the 1-nucleolar marker was used. Diploid embryos with only one nucleolus present were found following injection of unbroken erythroblasts into enucleated eggs from 2nu females. Triploid 2nu embryos were detected following injection of (diploid) 1nu erythroblasts into nonenucleated eggs from 2nu females. The most advanced development stages reached by these embryos did not, however, differ from the best results found in the first class of experiments: Nuclei from erythroblasts injected undamaged into nucleated or enucleated eggs never developed into a normal tadpole. Serial transfer experiments were performed using normally gastrulating embryos which had developed, following the injection of 1nu unbroken erythroblasts into recipient eggs. These donors for serial transfer experiments were checked for the presence of the 1nu marker. In addition they had passed through a normally cleaving eight-cell stage. No improvement in developmental capacity as compared to first transfer experiments could be found.     

Cyclic changes in amphibian egg microvilli occur during the division cycle: implication of MPF.

The microvilli (MV) of Pleurodeles (amphibian) eggs were examined following fertilization and compared with those of artificially activated eggs and enucleated eggs using scanning and transmission electron microscopy. The MV pattern in fertilized eggs was found to undergo a cyclic transformation during the course of the first few division cycles. Similar changes also occurred in the MV of artificially activated eggs and enucleated eggs. The reorganization of the MV was sensitive to cycloheximide and cytochalasin B, but was unaffected by colchicine. Thus, this MV alteration requires protein synthesis and microfilaments but microtubules are not implicated in this process. In addition, the effects on the MV pattern of the maturation or mitosis promoting factor (MPF) were tested. Injection of MPF into eggs at different times during the first division cycle nearly always induced an elongation of the MV. This observation suggests that MPF could regulate either directly or indirectly, via a MPF-sensitive factor, the cyclic transformation of amphibian egg MV.     

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.     

The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs.

Cleavage furrows of amphibian eggs exhibit characteristic morphological features: the presence of finger-like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin-streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of interspecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined with in vivo development in ligated rabbit oviduct, achieved higher blastoeyst development than in vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (     

Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation

The exposure of mouse zygotes pre-stained with Hoechst 33342 to u.v. irradiation for 20-30 sec significantly or completely inhibited development to blastocysts in vitro. However, development to the blastocyst stage of enucleated eggs receiving pronuclei from untreated eggs was as good as that of control reconstituted eggs when the cytoplasm originated from eggs exposed to u.v. irradiation for 20-30 sec, but was significantly lower when the cytoplasm was from eggs exposed for 40 sec. The chromosomes at the second metaphase stage could be removed with 15 sec of exposure to u.v. irradiation under a fluorescence microscope. Most eggs enucleated at the second metaphase that received a single inner cell mass nucleus (75%) showed pronuclear formation 6 h after activation; 23% of them developed to morphologically normal 2-cell eggs and 5% developed to blastocysts. These results demonstrate that the cytoplasm of mouse zygotes is more resistant to u.v. irradiation after Hoechst staining. Eggs at the second metaphase, from which chromosomes have been removed under a fluorescence microscope, can therefore be used as cytoplasm recipients for nuclear transplantation of inner cell mass nuclei.     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of intenpecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined within vim development in ligated rabbit oviduct, achieved higher blastocyst development thanin vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the intenpecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not speciesspecific; (ii) there is compatibility between intenpecies somatic nucleus and ooplasm during early development of the reconstructed egg.     

Lipovitellins and phosvitins of the fertilized eggs during embryo growth in the oviparous lizard Podarcis sicula

In the lizard Podarcis sicula, the major vitellogenin (VTG)-derived yolk proteins, lipovitellins and phosvitins, were extracted from the yolk globules of laid and fertilized eggs at different periods of incubation up to 44 days close to hatching. Embryonic development was almost over at this time. Yolk proteins were isolated by precipitation in saturated (NH(4))(2)SO(4), separated on SDS-PAGE and detected by Western blotting with homologous polyclonal anti/VTG antibody. Two lipovitellins of 110 and 116 kDa were always present in the yolk of laid eggs after 1, 10, 18, and 44 days from oviposition. Both these proteins were glycosylated and were recognized by the anti/VTG antibody; their N-terminal sequences were analyzed. Four phosvitins were detected in freshly laid eggs, but their number decreased during incubation, and after 44 days only a single protein of approximately 6.5 kDa was present. The results indicated that, in this lizard, during embryonic development, lipovitellins remain unchanged, whereas the phosphorylated components of yolk undergo continuous degradation.     

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos

As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. ³H-tryptophan or ³H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of ³H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( ) of the nuclei were densely labelled. Significant accumulation of ³H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae ; gastrulae ). Controls, involving RNase or boiling TCA, demonstrated that the ³H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with ³H-tryptophan or ³H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with ³H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones.In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.     

Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs

In this paper, we describe the procedure of large-scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30-mm length, 1-mm width, and 2-mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 μsec twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD-1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm. The proportion of development to blastocyst in the eggs fused with the pronuclear karyoplast derived from F1 (75 and 71%) was twice that of the eggs fused with the pronuclei derived from CD-1 strain (25 and 37%). After transfer to recipients, live young were obtained from both the eggs fused with karyoplast following one or two electrofusion exposures.     

Schistosoma japonicum: excretory-secretory products of the eggs during miracidial development.

The release pattern of excretory-secretory (E-S) products of Schistosoma japonicum eggs was investigated using eggs cultured in a chemically defined medium (MEMSE-J) for 16 days. The amount of protein released in culture supernatants was greater in 0- to 4-day and 12- to 16-day cultures than in 4- to 12-day cultures. The protein composition of E-S products and soluble extracts of newly laid eggs (N-SEA) and in vitro matured eggs (M-SEA) was analyzed by SDS-PAGE. Silver staining patterns of N-SEA and M-SEA were found to be similar except for the band at approximately 66 kDa, which appeared in highest concentrations in N-SEA. Western blot analysis with human infected sera showed antibody recognition of a 140- to 160-kDa antigen present in E-S products from mature eggs, while E-S products from immature eggs were unreactive. When either [35S]methionine or [3H]glucosamine was added to the culture medium, newly synthesized proteins or glycoproteins of the SEA and E-S products were labeled. Incorporation of both isotopes into SEA appears to correlate with developmental activity of the eggs. In contrast, release of E-S proteins and glycoproteins is more apparent as the miracidium matures. These results suggested that the source of E-S products from immature eggs is likely to be the collapsing vitelline cells and that of E-S products from mature eggs to be mainly miracidial secretions.     

Effect of egg composition on the developmental capacity of androgenetic mouse embryos.

Analysis of the developmental capacities of androgenetic and gynogenetic mouse embryos (bearing two paternal or two maternal pronuclei, respectively) revealed a defect in blastocyst formation of androgenetic, but not gynogenetic, embryos that was a function of the maternal genotype. Androgenetic embryos constructed using fertilized eggs from C57BL/6 or (B6D2)F1 mice developed to the blastocyst stage at frequencies similar to those previously reported, whereas androgenetic embryos constructed with fertilized eggs from DBA/2 mice developed poorly, the majority failing to progress beyond the 16-cell stage and unable to form a blastocoel-like cavity, regardless of whether the male pronuclei were of C57BL6 or DBA/2 origin. This impaired development was observed even in androgenetic embryos constructed by transplanting two male pronuclei from fertilized DBA/2 eggs to enucleated C57BL/6 eggs, indicating that the defect cannot be explained as the lack of some essential component in the DBA/2 cytoplasm that might otherwise compensate for androgeny. Rather, the DBA/2 egg cytoplasm apparently modifies the incoming male pronuclei differently than does C57BL/6 egg cytoplasm. Several specific alterations in the protein synthesis pattern of DBA/2 androgenones were observed that reflect a defect in the regulatory mechanisms that normally modulate the synthesis of these proteins between the 8-cell and blastocyst stages. These results are consistent with a model in which cytoplasmic factors present in the egg direct a strain-dependent modification of paternal genome function in response to epigenetic modifications (genomic imprinting) established during gametogenesis and indicate that preimplantation development can be affected by these modifications at both the morphological and biochemical levels.     

Transplantation of somatic nuclei into activated teleost eggs (the example of the loach, Misgurnus fossilis L.)]

Some peculiarities of the technique of nuclear transplantation for teleostean fishes are described, the loach (Misgurnus fossilis) taken as an example. The cells of the late high blastula were used as donors and the activated non-enucleated and enucleated (by X-rays, 20 kR) eggs of the loach as recipients. Following the nuclear transplantation in the activated non-enucleated eggs, 33 (out of 128) normal blastulae were obtained, one of which developed until hatching. Following the nuclear transplantation in the activated enucleated eggs, 34 (out of 251) blastulae were obtained, 6 embryos hatched and 2 larvae attained the stage of active feeding.     

山羊(Capra hircus)重构胚与再重构胚早期发育的研究

选择山羊8细胞期到桑椹胚期的正常胚胎或重构胚的卵裂球,或选择胚泡期胚胎的内细胞团细胞作为供核,并以LRH注射后26—28h的去核成熟卵球作为受体,制备重构胚或再重构胚,琼脂糖包埋,植入山羊输卵管内,体内培养4—6天发育结果表明:不同发育时期(8细胞期至胚泡期)的正常胚胎细胞核或重构胚细胞核中,至少有部分细胞核均保留着发育的全能性,这些细胞核在母系基因表达产物的调控下,实现重新编程,启动并完成正常发育的全过程。     

Transplantation of Nuclei from Lymphocytes of Adult Frogs into Enucleated Eggs

The technique of transplantation of nuclei from adult lymphocytes into enucleated eggs from Xenopus laevis (South African clawed toad) is described. The splenic lymphocytes from the one nucleolus mutant were bound via their immunoglobulin receptors to nylon fibers, derivatized with the antigen used for immunization. A technique for coupling other cell types with Woodward reagent is also described. The cells were broken by aspiration into a micropipette and injected into enucleated eggs. The egg pronucleus was eliminated by UV treatment followed by surgical removal. The origin of the genome of developing embryos was determined on karyotype preparations by looking for the nucleolar organizer on the chromosome pair No. 12. Participation of egg pronucleus in development was frequent as judged by the incidence of gynogenetic diploid individuals and of tetraploid animals exhibiting characteristics of both recipient egg and somatic cell donor karyotypes. Apparently normal, lymphocyte-derived tadpoles were obtained, but they died at 12 days. This shows that differentiated cells from adults, such as lymphocytes, can re-express the genes necessary for ontogeny.