Testicular and ovarian gonocytes from 20-day incubated chicken embryos contribute to germline lineage after transfer into bloodstream of recipient embryos

The present study was conducted to elucidate whether testicular and ovarian gonocytes obtained from 20-day incubated chicken embryos (stage 45) have the ability to migrate to the germinal ridges and contribute to germline lineage after transfer into the bloodstream of recipient embryos. Testicular and ovarian gonocytes were first identified as relatively large cells in a population of gonadal cells. The proportions of testicular and ovarian gonocytes in the total gonadal cells were 0.94 and 0.75% respectively, recognised as chicken vasa homologue-positive cells. Then, the dissociated gonadal cells obtained from 20-day incubated embryos containing testicular or ovarian gonocytes, with or without transfection, were transferred into recipient embryos. Expression of the introduced GFP gene was observed in the gonads of 6.5-day cultured recipient embryos (stage 30) in males and females, suggesting that the transferred testicular and ovarian gonocytes have the ability to migrate to the germinal ridges and enter the gonads. Furthermore, the presence of the donor-derived DNA was detected in the gonads of 20-day cultured recipient embryos in males and females, and also in the sperm samples obtained from the hatched male putative chimaeric chickens, suggesting that the transferred testicular and ovarian gonocytes were incorporated into the germline of chimaeric embryos and chickens. It is concluded that testicular and ovarian gonocytes obtained from 20-day incubated embryos have the ability to migrate to the germinal ridges after transfer into the bloodstream of recipient embryos, enter the gonads and contribute to the germline lineage of chimaeric embryos and chickens.     

Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells

Primordial germ cells differentiate into germ cells and have the ability to reacquire totipotency. Mouse primordial germ cells are identified by alkaline phosphatase staining of the extraembryonic mesoderm, and they proliferate and migrate to reach the genital ridges. Mouse primordial germ cells have never been maintained in culture exclusively for longer than a week without differentiation or dedifferentiation. Moreover, primordial germ cells have not been proliferated with urogenital complexes in vitro, because gonad culture has never been successful. It was thought that primordial germ cells could proliferate in a culture of urogenital complex under modified medium conditions resembling those in vivo; however, organ culture of mouse gonad has been performed with fetal calf serum or equine serum, and those sera produce conditions different from those in vivo. Therefore, mouse urogenital complexes were cultured in media containing rodent sera. As a result, it was possible to proliferate primordial germ cell-like cells outside gonads, and these cells very closely resembled primordial germ cells. In addition, motile primordial germ cell-like cells could be obtained. The ability to maintain primordial germ cell-like cells in culture by this intra-species culture method is important in the study of gametogenesis. Furthermore, this method is useful as a source of stem cells such as embryonic germ cells.     

In vitro development of mouse fetal germ cells into mature oocytes

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish an in vitro experimental model that allows one to study such mechanisms. Mouse follicular development has been studied in vitro over the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were cultured in vitro for 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were matured in vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula-blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro.     

Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated gamma-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.     

Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts' outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions. To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney. We conclude that somatic cells (FFs) can proliferate in early embryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.     

An extreme bias in the germ line of XY C57BL/6<->XY FVB/N chimaeric mice

Chimaeric analysis is a powerful method to address questions about the cell-autonomous nature of defects in spermatogenesis. Symplastic spermatids (sys) mice have a recessive mutation that causes male sterility due to an arrest in germ-cell development during spermiogenesis. Chimaeric mice were generated by aggregation of eight-cell embryos from sys (FVB/N genetic background) and wild-type C57BL/6 (B6) mice to determine whether the male germ-cell defect is cell-autonomous. The resulting FVB/N<->B6 chimaeras (<-> denotes fusion of embryos) were mated with FVB/N mice and coat colour of offspring was used to identify transmission of FVB/N or B6 gametes. Regardless of the relative contribution of B6 to somatic tissues of the chimaeras, almost all (282 of 284; 99.3%) offspring of B6 XY<->XY FVB/N (+/+ or sys/+) males (n = 9) received a FVB/N-derived paternal gamete. After mating of female B6<->FVB/N chimaeras, 51 of 73 (69.9%) offspring received an FVB-derived maternal gamete. Southern blot analysis of different tissues from chimaeric males indicated that, despite the presence of balanced chimaerism in somatic tissues, the germ line in B6 XY<->XY FVB/N mice was essentially FVB/N in composition. Thus there is a strong selective advantage for FVB/N male germ cells over B6 male germ cells in B6<->FVB/N-aggregation chimaeras at some stage during development of the male germ line. Each of three male chimaeras that were either B6 XY<->XY FVB/N (sys/sys) or B6 XX<->XY FVB/N (sys/sys) in composition was sterile, and testis histology was essentially sysmutant. This finding indicates that the function of the gene(s) affected in the sys mutation may be required in the testis, although whether expression is required in germ cells, somatic cells or both remains unknown. The extreme bias in transmission of male gametes has implications for experimental design in studies that use chimaeric analysis to address questions regarding the cell-autonomous nature of germ-cell defects in mice.     

Cats cloned from fetal and adult somatic cells by nuclear transfer

This work was undertaken in order to study the developmental competence of nuclear transfer (NT) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.     

Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI)

The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modified Eagle's medium. Morphologically normal early blastocysts were transferred to the uteri of recipient mares. Nuclear maturation rates and the rates of cleavage to the two-cell stage for injected oocytes were similar in the groups of oocytes that were matured in TCM-199 (49 and 63%), in co-culture with oviduct epithelial cells (53 and 65%) or in co-culture with fetal fibroblasts (51 and 57%). There were no significant differences in the proportions of blastocysts that developed from the two-cell embryos derived from oocytes matured by co-culture with either oviduct epithelial cells (30%) or fetal fibroblasts (17%). However, significantly higher proportions of blastocysts were produced from both these co-culture groups than from the groups of oocytes matured in TCM-199 only (P < 0.05). Six of the blastocysts that had developed from oocytes co-cultured with oviduct epithelial cells were transferred into recipient mares and four pregnancies resulted. These results demonstrate a beneficial influence of co-culture with either oviduct epithelial cells or fetal fibroblasts for maturation of oocytes in vitro.     

Survival and normal function of glycolysis-deficient mouse oocytes

Mouse embryos homozygous for a null allele of Gpi1 which encodes the glycolytic enzyme glucose phosphate isomerase fail to complete gastrulation and die at about embryonic day 7.5, but mutant cells can survive in fetal chimaeras in which they are mixed with wild-type cells. An adult female mouse chimaera, composed of wild-type cells and homozygous Gpi1(-/-) null mutant cells, was produced to test whether the presence of wild-type cells in the ovary allowed mutant oocytes to survive and function. This mouse produced 28 offspring, eight of which were derived from homozygous Gpi1(-/-) null oocytes. DNA in situ hybridization also showed that some Gpi1(-/-) follicle cells were able to survive in chimaeric ovarian follicles. It is likely that the survival of mutant follicle cells and fully functional mutant oocytes was mediated by the presence of wild-type cells that could provide metabolic intermediates and so bypass the block in the glycolytic pathway. Wild-type cumulus cells probably supported the growing GPI-deficient oocytes via metabolic co-operation, by passing ATP and other glycolytic products through gap junctions. It was concluded that female mouse germ cells and ovarian follicle cells do not need an intact endogenous glycolytic pathway if they can obtain appropriate metabolites from an exogenous source.     

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.     

Estrous synchronization and establishment of pregnancy in bovine embryo transfer recipients

Characteristics of donors, embryos, recipients, and transfer procedures were examined for relationships with establishment of pregnancy. Data were from records of 1202 embryos transferred by the professional staff of a large embryo transfer company. Average pregnancy rate was 74.6%. Variables affecting establishment of pregnancy were service sire, embryo quality, stage of estrous cycle in recipient, quadratic effect of synchrony, and transfer quality. Because the direct or indirect effect of sire must be established at conception, these data demonstrate a latent effect of sire on pregnancy. One would expect the highest pregnancy rate among excellent quality embryos transferred with no delays or complications into recipients that had displayed estrus at the same time or prior to the donor yet were relatively early in their estrous cycles. There also was a tendency for pregnancy rate to decrease with advancing maturity of the embryo.     

Successful transplantation of bovine testicular cells to heterologous recipients

While heterologous germ cell transplantation was successful in pigs and goats, autologous transplantation alone has been reported to result in donor-derived spermatogenesis in cattle. The objective of this study was to investigate whether the transplantation of heterologous germ cells could result in colonization of recipient testes in cattle of different breeds. Testicular cells were isolated from 8 Bos taurus donor bull calves and then transferred into 15 Bos indicus-cross bull calves. All animals were prepubertal, donors were aged 5-7 months and recipients 5-11 months, and scrotal circumferences ranged from 15 to 22 cm. Single cell suspensions of donor testicular cells, prepared by enzymatic digestion, were labelled with fluorescent dyes PKH26 or CFDA-SE, before transfer into the rete testis of recipients under ultrasonographic guidance. To assess the longevity of colonization by donor cells, recipients were castrated 2-30 weeks after cell transfer. Donor cells were observed in 15/25 (60%) of the testes that received PKH26-labelled cells, whereas no CFDA-SE-positive cell was identified in any recipients. The maturity of the donors or recipients (measured by scrotal circumference) did not affect colonization potential. In freshly isolated tubules, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the tubules. In frozen sections, PKH26-positive cells were identified on the seminiferous tubule basement membrane, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be spermatogonia. We conclude that PKH26 was more suitable for labelling donor testis cells and donor cells can be identified up to 6 months following transfer. These results indicate that allogeneic transplantation of testicular cells can occur between Bos taurus and Bos indicus cattle. Further studies will investigate functionality of transferred testicular cells.     

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7-19 of gestation.     

Influence of donor age on development of gonadal tissue from pouch young of the tammar wallaby, Macropus eugenii, after cryopreservation and xenografting into mice

Ovaries from a marsupial, the tammar wallaby (Macropus eugenii), were grafted into a eutherian recipient at known stages of development to ascertain whether normal development would occur. Xenografted ovaries from pouch young < 20 days old, before the onset of meiosis, retained few germ cells and developed tubule-like structures reminiscent of seminiferous cords. Ovaries from 50-day-old pouch young, which contain primordial follicles, developed into antral follicles and corpora lutea within the eutherian host, and produced hormones that stimulated the reproductive tract of the host. The timing of onset of antrum formation and the progress of follicle development were advanced relative to the timing of events in ovaries in situ. Frozen-thawed ovaries from 50-day-old donors developed into preantral follicles, but at a reduced rate and number. This finding shows that gonads of a marsupial species can develop as xenografts in a eutherian, forming large antral follicles. Accelerated follicular development in xenografts provides a potentially valuable model for studying the factors that control follicle development. Assisted reproduction of endangered marsupials may also be feasible using follicles from pouch young grown as xenografts in a eutherian host.     

Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply

Proliferation and partial meiotic maturation of germ cells in fetal ovaries is believed to establish a finite, non-renewable pool of primordial follicles at birth. The supply of primordial follicles in postnatal life should be depleted during folliculogenesis, either undergoing atresia or surviving to ovulation. Recent studies of mouse ovaries propose that intra- and extraovarian germline stem cells replenish oocytes and form new primordial follicles. We quantified all healthy follicles in C57BL/6 mouse ovaries from day 1 to 200 using unbiased stereological methods, immunolabelling of oocyte meiosis (germ cell nuclear antigen (GCNA)) and ovarian cell proliferation (proliferating cell nuclear antigen (PCNA)) and electronmicroscopy. Day 1 ovaries contained 7924+/-1564 (s.e.m.) oocytes or primordial follicles, declining on day 7 to 1987+/-203, with 200-800 oocytes ejected from individual ovaries on that day and day 12. Discarded oocytes and those subjacent to the surface epithelium were GCNA-positive indicating their incomplete meiotic maturation. From day 7 to 100 mean numbers of primordial follicles per ovary were not significantly depleted but declined at 200 days to 254+/-71. Mean numbers of all healthy follicles per ovary were not significantly different from day 7 to 100 (range 2332+/-349-3007+/-322). Primordial follicle oocytes were PCNA-negative. Occasional unidentified cells were PCNA-positive with mitotic figures observed in the cortex of day 1 and 12 ovaries. Although we found no evidence for ovarian germline stem cells, our data support the hypothesis of postnatal follicle renewal in postnatal and adult ovaries of C57BL/6 mice.     

The proteasomal inhibitor MG132 increases the efficiency of mouse embryo production after cloning by electrofusion

In this study, we cloned mice from ES cells by a post-electrofusion MG132 treatment and improved development of cloned embryos with a sequential cultivation protocol. When 5 microM MG132, a proteasome inhibitor, were used to treat the reconstructed embryos, the capacity of in vitro development, implantation and full-term development were significantly improved. Blastocyst formation rates of the reconstructed embryos from X4 ES cells (F1 strain derived from C57BL/6 x 129sv) and J1 ES cells obtained with or without MG132 treatment were 66.9% and 26.6%, and 66.1% and 34.5% respectively (P < 0.05). A total of 146 two-cell embryos cloned from X4 ES cells with MG132 treatment were transferred to recipients, and five cloned pups (3.4%) were born, of which four survived. When the same numbers of two-cell embryos cloned from X4 ES cells without MG132 treatment were transferred, however, no live-born mice were obtained. When embryos cloned from J1 ES cells without MG132 treatment were cultured in KSOM medium for 54 h followed by culture in CZB medium containing 5.6 mM glucose for 42 h, the blastocyst rate was significantly higher than when they were cultured in KSOM continuously for 96 h (34.5% vs 17.1%). However, sequential cultivation did not improve the development of embryos cloned with MG132 treatment and that of parthenotes. In conclusion, MG132 treatment increased the developmental potential of reconstructed mouse embryos, and sequential cultivation improved development of the embryos cloned by electrofusion without MG132 treatment.     

Evolution of the germ line-soma relationship in vertebrate embryos

The germ line and soma together maintain genetic lineages from generation to generation: the germ line passes genetic information between generations; the soma is the vehicle for germ line transmission, and is shaped by natural selection. The germ line and somatic lineages arise simultaneously in early embryos, but how their development is related depends on how primordial germ cells (PGC) are specified. PGCs are specified by one of two means. Epigenesis describes the induction of PGCs from pluripotent cells by signals from surrounding somatic tissues. In contrast, PGCs in many species are specified cell-autonomously by maternally derived molecules, known as germ plasm, and this is called preformation. Germ plasm inhibits signaling to PGCs; thus, they are specified cell-autonomously. Germ plasm evolved independently in many animal lineages, suggesting convergent evolution, and therefore it would be expected to convey a selective advantage. But, what this is remains unknown. We propose that the selective advantage that drives the emergence of germ plasm in vertebrates is the disengagement of germ line specification from somatic influences. This liberates the evolution of gene regulatory networks (GRNs) that govern somatic development, and thereby enhances species evolvability, a well-recognized selective advantage. We cite recent evidence showing that frog embryos, which contain germ plasm, have modified GRNs that are not conserved in axolotls, which represent more basal amphibians and employ epigenesis. We also present the correlation of preformation with enhanced species radiations, and we discuss the mutually exclusive trajectories influenced by germ plasm or pluripotency, which shaped chordate evolution.     

Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.     

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.