Parthenogenesis is a form of asexual reproduction found in females, where growth and development of embryos occurs without fertilization by a male. In plants, parthenogenesis means development of an embryo from an unfertilized egg cell, and is a component process of apomixis. The offspring produced by parthenogenesis are always female in species that use the XY sex-determination system, and male in those that use the ZW sex-determination system. Parthenogenesis (<Gr. “virgin birth”) is production of offspring by a female with no genetic contribution from a male and without meiotic chromosome reduction. The process is common reproductive strategy among insects such as aphids, flies, ants, and honeybees, but is also known to occur in vertebrates including lizards, snakes, fish, birds, and amphibians.
The first demonstration of artificially-stimulated parthenogenesis in vitro was made by Jacques Loeb (1899), who was able to activate oocytes from sea urchins and frogs by pricking them with a needle or by changing the ambient salt concentration. Pincus (1939) demonstrated parthenogenetic activation of mammalian eggs using temperature and chemical stimuli. Thus far, parthenogenetic activation of eggs has been studied in a variety of mammals including mice, goats, cows, monkeys, and humans. Plachot et al. described parthenogenesis in humans by examining 800 human oocytes and showed that 12 activated parthenogenetically and four underwent normal cleavage. Although there have been no reports of naturally-occurring human parthenotes, a human parthenogenetic chimera has been described. The juvenile patient presented with developmental delay, apparent sex reversal, and entirely parthenogenetic blood leukocytes. This finding confirmed the viability of chimeras in higher mammals as presaged by successful murine experiments over the previous two decades
Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications.
There is no confirmed example of de novo mammalian parthenogenetic reproduction, but mammalian oocytes can be artificially induced to undergo parthenogenesis in vitro by a two-step protocol involving electroporation and/or treatment with a chemical agent (ionomycin, ethanol, or inositol 1,4,5-triphosphate) to elevate Ca2+ levels transiently, followed by application of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-dimethylaminopurine). Success rates and viability appear to be organism dependent. Mouse parthenotes are capable of developing beyond the post-implantation stage in vivo; porcine parthenotes have developed up to post-activation day 29 (limb bud stage, past the early heart beating stage); rabbit parthenotes until day 10–11; primates (Callithrix jacchus) have only been shown to implant. The reason for this arrested development is believed to be due to genetic imprinting. In normal zygotes maternal and paternal haploid genomes are epigenetically distinct, and both sets are required for successful development. Indeed, unstable chromosome modifications in the form of DNA methylation or histone modification are distinctly different in human sperm, compared to eggs. Therefore each gamete carries unique patterns of gene expression into the embryo. Since all genetic material in parthenotes is of maternal origin, there is no paternal imprinting component and this prevents proper development of extraembryonic tissues whose expression is regulated by the male genome. In most mammals – including primates – oocytes are arrested at metaphase II just before ovulation. Cytogenetic microscopy shows the presence of a 2n polar body under the zona pellucida and a 2n protonucleus in the cytoplasm. After chemical activation to mimic the effects of sperm penetration on changes in cellular Ca2+ gradient, the cell fails to complete meiosis II. Instead, the second polar body is never extruded, resulting in a diploid protonucleus derived from two sets of sister chromatids. These chromatids then begin to undergo mitosis resulting in a parthenote manifesting uniparental disomy.
Although the derivation of embryonic-like stem cells from oocytes (parthenogenetic stem cells, PSC) is relatively inefficient (perhaps due to complexities of genomic imprinting), when they are differentiated into adult tissues, they appear fully functional.
In spite of non-viability of monkey parthenotes, the extracted stem cells seem to assume the morphology and functional behavior of HESC and express appropriate ESC markers. They have embryonic-like replicative ability and have been propagated in vitro in an undifferentiated state for up to 14 months. In vitro, they have been differentiated into cardiomyocyte-like cells, smooth muscle, beating ciliated epithelia, adipocytes, several types of epithelial cells, as well as dopaminergic and serotoninergic neurons. Almost all of these neurons express TUJ1 (beta-tubulin III), and up to 25% of the TUJ1+ cells co-express tyrosine-hydroxylase. This latter enzyme marker is considered diagnostic for catecholaminergic neurons (dopamine, norepinephrine, and epinephrine. Furthermore, HPLC analysis of culture media following a depolarizing KCl-buffer identifies the release of the neurotransmitters dopamine and serotonin from the cells. After two weeks of differentiation, about half of the cells demonstrate neuronal morphology and begin to express voltage-dependent sodium channels that can be blocked by tetrodotoxin.
These observations are recapitulated in vivo, since injection of monkey PSC into immunocompromised mice induces formation of benign teratomas containing tissue derivatives from all three germ layers (ectoderm, endoderm and mesoderm) including cartilage, muscle, bone, neurons, skin, hair follicles, and intestinal epithelia. Of particular note is the apparent tendency of these cells to differentiate into neuronal tissues, as has been noted by chimera studies. The reasons for this underlying preference are not well understood although one possible explanation is that it is a consequence of purely maternal genomic imprinting, reflecting a lack of epigenetic balance that would be conferred by paternally-imprinted genes.
To be sure, parthenotes are not free from ethical controversy and are viewed by some in society as artificial entities that in some sense represent ‘tampering with nature.’ Since a parthenote is analogous to a mature ovarian teratoma (a spontaneous in vivo tumorigenic event) the de facto acceptance of experiments using teratoma tumor tissue lends some legitimacy to experimentation on parthenotes. These contradictions await reconciliation in a comprehensive ethical framework.
