Recipient Eggs Research Articles

A newly developed cloning technique in sturgeons; an important step towards recovering endangered species

Several steps of sturgeon somatic cell nuclear transfer (SCNT) have been recently established, but improvements are needed to make it a feasible tool to preserve the natural populations of this group of endangered species. The donor cell position inside the recipient egg seems to be crucial for its reprogramming; therefore by injecting multiple donor somatic cells instead of a single cell with a single manipulation, we increased the potential for embryo development. Using the Russian sturgeon Acipenser gueldenstaedtii as a multiple cell donor and sterlet Acipenser ruthenus as the non-enucleated egg recipient, we obtained higher proportion of eggs developing into embryos than previously reported with single-SCNT. Molecular data showed the production of a specimen (0.8%) contained only the donor genome with no contribution from the recipient, while two specimens (1.6%) showed both recipient and donor genome. These findings are the first report of donor DNA integration into a sturgeon embryo after interspecific cloning. In all, we provide evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that the sturgeon cloning faces limitations, to date it is the most promising technique for their preservation.

Mitochondrial matters: Mitochondrial bottlenecks, self-assembling structures, and entrapment in the female germline

Mitochondrial replacement therapy, a procedure to generate embryos with the nuclear genome of a donor mother and the healthy mitochondria of a recipient egg, has recently emerged as a promising strategy to prevent transmission of devastating mitochondrial DNA diseases and infertility. The procedure may produce an embryo that is free of diseased mitochondria. A recent study addresses important fundamental questions about the mechanisms underlying maternal inheritance and translational questions regarding the transgenerational effectiveness of this promising therapeutic strategy. This review considers recent advances in our understanding of maternal inheritance of mitochondria, implications for fertility and mitochondrial disease, and potential roles for the Balbiani body, an ancient oocyte structure, in mitochondrial selection in oocytes, with emphasis on therapies to remedy mitochondrial disorders.

A Protocol for Adult Somatic Cell Nuclear Transfer in Medaka Fish (Oryzias latipes) with a High Rate of Viable Clone Formation

Previously, we successfully generated fully grown, cloned medaka (the Japanese rice fish, Oryzias latipes) using donor nuclei from primary culture cells of adult caudal fin tissue and nonenucleated recipient eggs that were heat shock-treated to induce diploidization of the nuclei. However, the mechanism of clone formation using this method is unknown, and the rate of adult clone formation is not high enough for studies in basic and applied sciences. To gain insight into the mechanism and increase the success rate of this method of clone formation, we tested two distinct nuclear transfer protocols. In one protocol, the timing of transfer of donor nuclei was changed, and in the other, the size of the donor cells was changed; each protocol was based on our original methodology. Ultimately, we obtained an unexpectedly high rate of adult clone formation using the protocol that differed with respect to the timing of donor nuclei transfer. Specifically, 17% of the transplants that developed to the blastula stage ultimately developed into adult clones. The success rate with this method was 13 times higher than that obtained using the original method. Analyses focusing on the reasons for this high success rate of clone formation will help to elucidate the mechanism of clone formation that occurs with this method.

Production of Somatic Chimera Chicks by Injection of Bone Marrow Cells into Recipient Blastoderms

Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells' (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.

Dissection of the maternal effects on puberty onset by embryo transplantation in mouse.

Puberty onset in mammals is affected by multiple genetic and environmental factors. Among which, the maternal effect could have played a considerable role. In our previous study, we found that the F1 offspring from reciprocal crosses between C3H/HeJ (C3H) and C57BL/6J (B6) mice differed significantly in the timing of puberty in both sexes, though they had identical genomic background. In order to dissect the causative factors to such phenomenon of maternal effect, embryos from reciprocal crosses of C3H/HeJ and C57BL/6J mice were collected and transplanted to the uterus of either strain of mothers, and the puberty onset of pups were compared between different recipient mothers and egg origins. The results showed that the male pups from C3H recipient mothers attained puberty onset earlier than those from B6 recipients significantly, while the female pups did not show such difference. On the other hand, the egg origin made no difference in the puberty onset of either sex, yet it influenced the birth weight of female pups significantly (p<0.05). The manipulation of embryo transplantation delayed the puberty onset of pups dramatically. A mitochondria substitution strain between B6 and C3H (BmC), which had the genome background of B6 and a mitochondrial hyplotype of C3H, had the same phenotype of puberty onset as B6. The integrated results indicated that the uterine environment was the major causative factors to the maternal effect on the differential puberty onset in reciprocal crosses of F1 hybrids between B6 and C3H mice.

Interspecific Duck–Chicken Chimeras

The production of quail–Peking duck, Peking duck–quail and Maya duck–chicken chimeras by transferring donor (quail, Peking duck and Maya duck) blastoderm cells into the subgerminal cavity of the recipient (Peking duck, quail and chicken) eggs is described. Production of chimeras by transferring blastoderm cells into the recipient embryo should provide a basis for the investigation of fertilisation barriers in interspecies germline chimeras. It might help to protect endangered birds, contribute to further understanding of physiology and embryonic development in poultry, and provide a technical method for poultry transgenics.

Fixing oocytes? A bovine model provides new hope

In a previous issue of Reproductive BioMedicine Online, Chiaratti and co-workers presented a bovine model for ooplasmic transfer, which demonstrated a positive effect on early development. Developmental deficits resulting from artificial treatment of recipient eggs with a toxic compound were ameliorated by the addition of small volumes of healthy donor cytoplasm. This model provides an important advance in the understanding of ooplasmic effects in early development and addresses issues about the prior human trials in this area.

Chromosome transfer in mature oocytes

In this article, we describe detailed protocols for the isolation and transfer of spindle-chromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindle-chromosomal complex is visualized using a polarized microscope and extracted into a membrane-enclosed karyoplast. Chromosomes are then reintroduced into an enucleated recipient egg (cytoplast), derived from another female, by karyoplast-cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA (mtDNA), from another female. This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy offspring. The protocol was initially developed for monkey oocytes but can also be used in other species, including mouse and human oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Chromosome transfer between the cohorts of oocytes isolated from two females can be completed within 2 h.

Enucleation of feeder cells and egg cells with psoralens

The cell nucleus must be inactivated or destroyed in order to generate feeder layers for cultured cells or to prepare recipient egg cells for nuclear transfer. Existing enucleation techniques are either cumbersome or employ toxic chemicals. Here we report a new method to enucleate cells by treatment with a psoralen and long-wave ultraviolet light. The technique is >90% efficient and causes little cytoplasmic damage to the treated cell. We have used psoralen treatment to enucleate a wide variety of cells, including eggs, sperm, HeLa cells, and fibroblasts. Colonies of human embryonic stem cells (hESCs) and human keratinocyte precursors grown on psoralen-treated feeders are indistinguishable from those grown on gamma-irradiated or mitomycin C-treated cells. Psoralen enucleation provides a rapid, simple, and non-toxic method to generate feeder cells. The technique is also useful for nuclear transfer studies in species with large eggs whose cleavage divisions are not regulated by cell-cycle checkpoints.

Offspring Sex in a Lizard Depends on Egg Size

Current paradigms may substantially underestimate the complexity of reptilian sex determination. In previous work, we have shown that the sex of a hatchling lizard (Bassiana duperreyi, Scincidae) does not depend entirely on its genes (XX versus XY sex chromosomes); instead, low nest temperatures can override genotype to produce XX as well as XY males. Our experimental studies now add a third mechanism to this list: sex determination via yolk allocation to the egg. Within each clutch, the eggs that produce daughters are larger than those that produce sons. If (and only if) eggs are incubated at low temperatures, removing yolk from a newly laid egg turns the offspring into a male. Adding yolk from a larger (but not smaller) egg turns the recipient egg's offspring into a female. Remarkably, then, offspring sex in this species is the end result of an interaction between three mechanisms: sex chromosomes, nest temperatures, and yolk allocation.

Antagonist protocol in oocyte-donors egg sharing programme is equally efficient as the agonist protocol in patients of same age group

OBJECTIVE: The study was undertaken to determine the efficacy of the antagonist protocol in a oocyte donor – recipient egg sharing programme and compare it with the agonist protocol used in regular infertility patients.DESIGN: Retrospective study involving oocyte donors and regular infertility patients comparing antagonist and agonist protocols during 1/10/2007 till 31/03/2008.MATERIALS AND METHODS: 32 women with proven fertility were recruited as oocyte donors (aged between 20 to 30years). Antagonist protocol was used for their ovarian stimulation (187.5 – 200 IU r-FSH from day 2; antagonist 0.25mg per day, flexible protocol). Recipients (n=70) were prepared with estradiol valerate and micronized progesterone. Every recipient received at least 7 mature eggs retrieved from the donors. 79 patients (aged between 20 to 30 years) underwent ovarian stimulation using agonist protocol (down regulation with Luprolide acetate 0.5 mg and ovarian stimulation with 200- 225 IU r-FSH). Three embryos were transferred on day-2 after oocyte collection. Pregnancy was defined as b hCG of ≥100 mIU/ ml 15 days after embryo transfer (ET).RESULTS: The average age (26.7 and 27.2 years), average number of eggs (18 and 15), % of mature eggs (90 and 87%), normal fertilization rate (77 % apiece), cleavage rate (97.5 and 98.7%), % useable embryos (97 and 96%) and four cell-grade1 embryos (53 and 56%) are comparable in the oocyte-donors (antagonist protocol) and the regular infertility patients (agonist protocol). The number of patients covered for embryo transfer (70 and 79), pregnancy rate per embryo transfer (34 and 32%), multiple pregnancy rate (29 % apiece) and the implantation rate per embryo (16.3 and 15%) are also not significantly different in oocyte- donor (antagonist protocol) and regular patients (agonist protocol). By sharing eggs from donors, 70 ET could be carried out from 32 donor stimulations [2.2 recipients / donor] giving a pregnancy rate of 75% per donor.CONCLUSIONS: This study shows that in the same age group the antagonist protocol yields almost similar results compared to agonist protocol. Further, sharing of eggs from donor helps twice the number of recipients per donor without compromising the pregnancy rate. OBJECTIVE: The study was undertaken to determine the efficacy of the antagonist protocol in a oocyte donor – recipient egg sharing programme and compare it with the agonist protocol used in regular infertility patients. DESIGN: Retrospective study involving oocyte donors and regular infertility patients comparing antagonist and agonist protocols during 1/10/2007 till 31/03/2008. MATERIALS AND METHODS: 32 women with proven fertility were recruited as oocyte donors (aged between 20 to 30years). Antagonist protocol was used for their ovarian stimulation (187.5 – 200 IU r-FSH from day 2; antagonist 0.25mg per day, flexible protocol). Recipients (n=70) were prepared with estradiol valerate and micronized progesterone. Every recipient received at least 7 mature eggs retrieved from the donors. 79 patients (aged between 20 to 30 years) underwent ovarian stimulation using agonist protocol (down regulation with Luprolide acetate 0.5 mg and ovarian stimulation with 200- 225 IU r-FSH). Three embryos were transferred on day-2 after oocyte collection. Pregnancy was defined as b hCG of ≥100 mIU/ ml 15 days after embryo transfer (ET). RESULTS: The average age (26.7 and 27.2 years), average number of eggs (18 and 15), % of mature eggs (90 and 87%), normal fertilization rate (77 % apiece), cleavage rate (97.5 and 98.7%), % useable embryos (97 and 96%) and four cell-grade1 embryos (53 and 56%) are comparable in the oocyte-donors (antagonist protocol) and the regular infertility patients (agonist protocol). The number of patients covered for embryo transfer (70 and 79), pregnancy rate per embryo transfer (34 and 32%), multiple pregnancy rate (29 % apiece) and the implantation rate per embryo (16.3 and 15%) are also not significantly different in oocyte- donor (antagonist protocol) and regular patients (agonist protocol). By sharing eggs from donors, 70 ET could be carried out from 32 donor stimulations [2.2 recipients / donor] giving a pregnancy rate of 75% per donor. CONCLUSIONS: This study shows that in the same age group the antagonist protocol yields almost similar results compared to agonist protocol. Further, sharing of eggs from donor helps twice the number of recipients per donor without compromising the pregnancy rate.

Morphometric characteristics and reproductive capacity of nuclear transplants derived from embryonic cells of loach, Misgurnus anguillicaudatus

Nuclear transplants of loach were produced by transplantating blastula cell nuclei into nonenucleated unfertilized eggs, using recipient eggs and donor cells distinguished by different polymorphic microsatellites. Of the total of 2,847 operated eggs, 143 hatched and 119 developed to the feeding larval stage. For 15 nuclear transplants (i.e., 11.1-year-old fish and 4.2-year-old fish) that survived up to the adulthood, DNA analysis and karyotype analysis were performed. Results showed that, of the 15 fish, 14 had only a nucleus derived from the donor; moreover, 12 were diploids, 1 was a triploid, 1 was a tetraploid, and 1 was a diploid-tetraploid mosaic with both donor and recipient nuclei. For the 12 fish with only a 2n donor nucleus, morphometric analysis was performed, and two female fish and two male fish were mated with normal fish. The fish with only a 2n donor nucleus were determined to be morphometrically identical to normal fish: they had normal gametogenesis and were able to reproduce. Currently, nuclear transplantation technology is beginning to be adopted in fisheries. Biological information on nuclear transplants obtained in this study can be used in the development of nuclear transplantation technology.

Novel method for the nuclear transfer of adult somatic cells in medaka fish (Oryzias latipes): Use of diploidized eggs as recipients

Until recently, the nuclear transfer of adult somatic cell nuclei in fish has been unsuccessful. This is primarily because of chromosomal aberrations in nuclear transplants, which are thought to arise due to asynchrony between the cell cycles of the recipient egg and donor nucleus. We recently succeeded in circumventing this difficulty by using a new nuclear transfer method in medaka fish (Oryzias latipes). Instead of enucleated eggs, the method uses non-enucleated and diploidized eggs, obtained by retention of the second polar body release, as recipients in the nuclear transfer of primary culture cells from the caudal fin of an adult green fluorescent protein gene (GFP)-transgenic strain. We found that 2.7% of the reconstructed embryos grew into diploid and fertile adults exhibiting donor expression characteristics and transmission of the GFP marker gene to progeny. The mechanism underlying the generation of nuclear transplants using this method is unknown at present; however, analyses of donor and recipient nuclei behavior and the cytoskeletal mechanisms involved in the early developmental stages, as well as the special ability of diploidized eggs to facilitate reprogramming of the donor nuclei will result in elucidation of the mechanism.

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

Generation of Fertile and Diploid Fish, Medaka (Oryzias latipes), from Nuclear Transplantation of Blastula and Four-Somite-Stage Embryonic Cells into Nonenucleated Unfertilized Eggs

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.

Production of Transgenic Chimeric Chickens Using Blastodermal Cells

A practical approach was proposed to produce transgenic chimeric chickens using blastodermal cells (BCs). The chicken BCs were mechanically dissociated and transferred into the recipient eggs that had been exposed to 500 rads irradiation of 6 0 Co and windowed on the equatorial plane. Chimeric chickens were generated using two models: the crosses (MXL) from Black Minors (ii,EE,b/b) ♂×Barred Leghorns (ii,ee,B/-) ♀ as donors and White Leghorns (WL, II) as acceptors (Model 1), or the Black Heifengs (BH, ii,EE,bb) as donors and Hua-xing white (HW, II) as recipients (Model 2). The treated eggs were incubated in their original shells in normal conditions until hatching. Green fluorescent protein (GFP) gene was transferred into the BCs derived from MXL and BH via lipofectamine and the pEGFP-C1, and transfection efficiency into the BCs was examined under a fluorescent microscope. Potential transgenic chimeras were selected based on the proposed methods in this study. Using the fresh BCs, the best rate of phenotypic chimeras was 6.7% and 26.0% in model-1 groups, and model-2 groups, respectively. We also described the optimized conditions for transfection. Although 30% of the BCs transfected in vitro emitted green light under an inverted fluorescent microscope, no embryos injected with the transfected BCs expressed foreign GFP gene at 3-4 days.

Paternal mitochondrial DNA transmission during nonhuman primate nuclear transfer.

Offspring produced by nuclear transfer (NT) have identical nuclear DNA (nDNA). However, mitochondrial DNA (mtDNA) inheritance could vary considerably. In sheep, homoplasmy is maintained since mtDNA is transmitted from the oocyte (recipient) only. In contrast, cattle are heteroplasmic, harboring a predominance of recipient mtDNA along with varying levels of donor mtDNA. We show that the two nonhuman primate Macaca mulatta offspring born by NT have mtDNA from three sources: (1) maternal mtDNA from the recipient egg, (2) maternal mtDNA from the egg contributing to the donor blastomere, and (3) paternal mtDNA from the sperm that fertilized the egg from which the donor blastomere was isolated. The introduction of foreign mtDNA into reconstructed recipient eggs has also been demonstrated in mice through pronuclear injection and in humans through cytoplasmic transfer. The mitochondrial triplasmy following M. mulatta NT reported here forces concerns regarding the parental origins of mtDNA in clinically reconstructed eggs. In addition, mtDNA heteroplasmy might result in the embryonic stem cell lines generated for experimental and therapeutic purposes ("therapeutic cloning").

Cytoplasmic transfer in the mouse in conjunction with intracytoplasmic sperm injection

Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been used clinically to facilitate human pregnancies. Data reported here describe the first characterization of CT coincident with intracytoplasmic sperm injection in the mouse system. Sibling oocytes were used to transfer 2, 4, or 6 pl of ooplasm to a recipient egg along with a sperm head using piezo-actuated injection. Survival and fertilization after CT were comparable to controls at 2 pl and 4 pl, but survival was significantly reduced with 6 pl volumes. Development to the blastocyst stage was also inversely related to CT volume, with some decline beginning with the 4 pl CT group. However, some blastocysts did develop in all of the groups. The results are in contrast with human eggs, which tolerate larger CT volumes. Results indicate that the mouse system can be used to characterize the transfer of exogenous materials concomitant with sperm injection, provided that the CT volume is not excessive.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo.

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%).

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the unclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation of gene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.