Biomedical subjects
Publications and source records attributed to P J Donovan.
Pluripotent stem cells can be expanded seemingly indefinitely in culture, maintain a normal karyotype and have the potential to generate any cell type in the body. As such they represent an incredible resource for the repair of diseased or damaged tissues in our bodies. These cells also promise to open a new window into the embryonic development of our species.
Somatic cells of whole Syrian hamster fetuses (gestation day 13) were isolated and tested by an in vivo/in vitro mutation assay for spontaneous mutation frequencies using independent 6-thioguanine (6-TG), diphtheria toxin (DT), and ouabain mutation selection systems. Optimum conditions were ascertained. For 6-TG mutants, a total of 21 mutants were found in cells from 24 litters on 1993 plates, for an overall mutant frequency of 1.8 x 10(-7) per viable cell with 12 positive litters. In all, 26 litters were tested using DT; 77 mutants were found in 840 plates, yielding an overall mutant frequency of 2.6 x 10(-7), with 20 positive litters. No correlations or familial effects were found among 23 litters tested for both DT and 6-TG. Of 14 litters which were tested for ouabain mutants, 4 were positive, with a total of 5 mutants found on 988 plates, for an overall mutant frequency of 7.6 x 10(-8). For 14 F344 rat fetuses, the overall 6-TG spontaneous mutation frequency was determined to be 1.6 x 10(-7). From the data, estimates of mutation rates were calculated. For mutation to 6-TG resistance the rate was 8.3 x 10(-8), for mutation to DT resistance the rate was 8.1 x 10(-8) and for ouabain, the spontaneous mutation rate was 5.7 x 10(-8). For F344 rat, the spontaneous mutation rate was 1.1 x 10(-7). Induced mutant frequencies after in utero exposure to 1 mmol/kg N-ethyl-N-nitrosourea (ENU) were 311, 135 and 200 times the spontaneous value for 6-TG, DT and ouabain, respectively, for Syrian hamster fetal cells and 125 times the spontaneous 6-TG value for fetal F344 rat cells. Both spontaneous mutation frequencies and underlying spontaneous mutation rates are low, consistent with the view that fetal cells exercise extremely tight control over DNA fidelity.
A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.
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Germ cells hold a unique place in the life cycle of animal species in that they are the cells that will carry the genome on to the next generation. In order to do this they must retain their DNA in a state in which it can be used to recapitulate embryonic development. In the normal life cycle, the germ cells are the only cells that retain this ability to recapitulate development, referred to as developmental totipotency. The molecular mechanisms regulating developmental potency are poorly understood. Recently its has been shown that germ cells can be turned into pluripotent stem cells when cultured in specific polypeptide growth factors that affect their survival and proliferation. The ability to manipulate developmental potency in germ cells with growth factors allows the underlying mechanisms to be dissected. Germ cells are also the only cells that undergo the unique reductive division of meiosis. This too is essential for the ability of germ cells to form the gametes that will carry the genome into the next generation. Arguably meiosis is the most important division in the life of a nascent organism. Defects in meiosis can result in embryonic or fetal loss or, if the animal survives, in the birth of an individual with chromosomal abnormalities. Recent advances in our understanding of meiosis have come from knockout mice and studies on genes identified through studies of human infertility. This review will focus on these two key aspects of germ cell biology, developmental potency and meiosis.
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The extremely high rate of cell division that occurs during early embryogenesis is hypothesized to predispose to high rates of mutation after chemical exposure. We tested this supposition experimentally. To probe the variation in susceptibility to mutation induction as a function of gestation stage, somatic cells of the developing Syrian hamster were isolated after transplacental treatment with N-ethyl-N-nitrosourea (ENU). Mutants were quantified using either 6-thioguanine (6-TG) or diphtheria toxin (DT) as selective agents. Several different approaches were used. In one, three litters were exposed on each gestation day and fetuses were removed on day 13. Maximum fetal sensitivity to ENU's genotoxic action was noted when treatment was at days 8 and 9, fewer mutants being obtained with earlier and later exposures. To compensate for the low numbers of target cells early in gestation, this experiment was repeated using larger numbers of litters exposed at the earlier time points, and the highest mutation frequency was now found to occur after treatment on gestation days 6 and 7. In the second approach, mutations were quantified in cells harvested 24 h after transplacental ENU exposure. Here again, embryos exposed at earlier times of gestation were more susceptible than those treated at later periods. Based on the total cell numbers in embryos and fetuses at each gestation day, we conclude that mutation frequency is maximal on day 6, corresponding to the primitive streak stage with extremely high rates of cell division.
In a clear demonstration of the changing sensitivity of the developing mammal to transplacental carcinogenesis, Ivankovic and Druckrey [S. Ivankovic, H. Druckrey, Transplacentare Erzeugung maligner Tumoren des Nervensystem: I. Athyl-nitroso-harnstoff (ANH) an BD IX-Ratten, Z. Krebsforsch. 71 (1968) 320-360] exposed pregnant BD IX rats to a pulse of N-ethyl-N-nitrosourea (ENU), a reactive carcinogen with a half-life of 20 min. No tumors were seen with ENU exposure before gestation day 12, but the multiplicity of neurogenic tumors increased steadily thereafter and was greatest with treatment on day 20, followed by a decline in sensitivity for the last three days of gestation. Similarly, a transplacental study of ENU in the Syrian hamster [B.A. Diwan, S. Rehm, J.M. Rice, Age- and dose-dependent transplacental carcinogenesis by N-nitrosoethylurea in Syrian golden hamsters, J. Cancer Res. Clin. Oncol. 122 (1996) 643-652] found that the numbers of tumors induced were greatest after exposure of late fetal stages. While these observations suggested that embryonic cells are refractory to carcinogenesis, an alternative explanation could be that a significant tumor yield was not observed because too few target cells were present in the embryo. I have resolved this issue by combining these published data with others on the numbers of neuroectodermal cells in the developing BD IX rat brain [R. Müller, M.F. Rajewsky, Elimination of O6-ethylguanine from the DNA of brain, liver, and other rat tissues exposed to ethylnitrosourea at different stages of prenatal development, Cancer Res. 43 (1983) 2897-2904] and total cell counts of successive developmental stages of the Syrian hamster fetus [P.J. Donovan, G.T. Smith, Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster, Mutation Res., 1999, this issue], allowing the risk per cell at different stages of gestation to be calculated. Sensitivity to carcinogenesis was found to be greatest early in gestation and to decrease as gestation proceeds. For the rat model, tumor frequency per cell changed from 1.3x10(-6) at day 12 exposure to 2.6x10(-8) at day 23 exposure, a 50-fold decrease. For the hamster model, the tumor-initiation rate decreased 1250-fold from 1.2x10(-5) at day 7 exposure to 9.6x10(-9) at day 13 exposure. Thus, two independent experiments with different rodent species demonstrate that sensitivity of individual cells to damage leading to transplacental carcinogenesis is greatest in the early fetus and lessens markedly as gestation proceeds, in parallel with changing sensitivity to mutation (Donovan et al., Mutat. Res., this issue).
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An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.
Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.
Leukemia inhibitory factor (LIF) stimulates the growth of primordial germ cells (PGCs) in mouse embryo. However, as neither mice lacking LIF nor mice lacking the LIF receptor show defects in PGC growth, an alternate cytokine for PGC growth has been postulated. We investigated the role of mouse oncostatin M (mOSM), which is structurally and functionally related to LIF, in germ cell development. While LIF enhanced the survival of migratory as well as postmigratory PGCs, mOSM acted only on the postmigratory PGCs. Consistent with its biological activity, mOSM was found to be expressed in developing gonads. In the male, Sertoli cells in neonatal testis express mOSM; however, its expression is downregulated in adult testes. Moreover, mOSM enhanced the proliferation of Sertoli cells derived from neonatal testes in vitro more than human OSM or LIF. In contrast, postnatal ovaries do not express mOSM. These results indicate that mOSM is a stage- and sex-specific autocrine growth factor for Sertoli cells.
During eukaryotic evolution, multicellular organisms have evolved multiple members of gene families that may display unique, partially overlapping, or redundant functions during development. More than 75% of the C. elegans genome has been sequenced, which represents approximately 95% of the coding sequences. This provides a unique opportunity to identify most, if not all, of the members of a given gene family. We have searched the C. elegans genome database for members of a key family of cell cycle regulators, the CDC25 phosphatases, and have identified four genes. The four C. elegans genes represent a larger family within a single organism than has been reported so far in Drosophila, mice and humans. An amino acid comparison revealed a high degree of similarity and identity within the phosphatase domain. This analysis also identified an expanded consensus sequence that can be used to discover new members of the CDC25 phosphatase family. However, the four C. elegans sequences display a few novel amino acid substitutions in the residues surrounding the invariant catalytic motif CX5R. These data demonstrate the value of genome database searching for identifying new members of known gene families, understanding genetic diversity, and for studying gene structure.
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The origin of hematopoietic stem cells (HSCs) and their growth factor requirement are poorly understood. Here we describe a new in vitro culture system of the aorta-gonad-mesonephros (AGM) region, where long-term repopulating HSCs first arise. We demonstrate that oncostatin M (OSM) is expressed in the AGM and is absolutely required for the expansion of multipotential hematopoietic progenitors in vitro. In addition, OSM enhances the formation of endothelial cell clusters. Thus, OSM appears to be a key cytokine for the development of multipotential hematopoietic progenitors in the AGM, possibly acting on common precursor cells between HSCs and endothelial cells. By using the AGM culture derived from macrophage colony-stimulating factor (M-CSF)-deficient op/op mutant embryos, we also show a pivotal role for M-CSF in fetal myelopoiesis.
Primordial germ cells (PGCs) are the embryonic progenitors of mature germ cells. During their proliferative stage, murine PGCs may be transiently cultured on mitotically inactive feeder layers. This culture system has permitted identification of several growth factors active toward PGCs. We and others have previously identified basic fibroblast growth factor (bFGF) as a powerful mitogen in this system. Here we characterize some of the functions of bFGF in PGC culture. Our data demonstrate that fibroblast growth factor (FGF) receptors I and II are present in the developing gonad and are consistent with expression of these receptors by PGCs. Moreover, PGCs can bind radiolabeled bFGF in vitro, demonstrating that the factor can act directly on these cells. While mitotic PGCs of either sex are shown to bind radiolabeled bFGF, oogonia that are undergoing meiotic arrest exhibit reduced bFGF binding, indicating potential developmental regulation of an FGF receptor.