Ultrastructural analysis reveals abnormal mitochondria in cloned blastocysts

Somatic cell nuclear transfer (SCNT) is a powerful technique, but still very inefficient despite 20 years passed by since the cloned mammal was born. We have recently shown that the major cause of abnormalities observed in cloned fetuses are mitochondrial dysfunctions in placenta collected from cloned sheep. Investigations on mitochondria in SCNT are limited to the mtDNA hetero/homoplasmy in cloned offspring, whereas no data is available for an eventual role of mitochondria dysfunction on the developmental failure of cloned animals. Here we wanted to know whether mitochondrial abnormalities are observed already in cloned blastocysts since mitochondrial replication does not occur after the hatched blastocysts stage. SCNT and in vitro processed (IVP) blastocysts were produced and analysed for mitochondrial structure and functionality. First, embryos were analysed using transmission electron microscope (TEM). Drastic differences in mitochondrial structure between SCNT and IVP blastocysts were observed. Decrease density of mature mitochondria, very high degree of cytoplasmic vacuolisation, numerous cytoplasmic vesicle and autophagosomes were observed in SCNT blastocysts. Moreover, statistically lower expression of major mitochondrial, autophagic and apoptotic proteins were observed in SCNT embryos. Obtained results clearly shown that mitochondrial abnormalities are already observed in blastocysts stage embryos. It is important to point out that activity of mitochondria are strictly control by nuclear signals, thus, obtained results may suggest that incomplete nuclear reprogramming in cloned nucleus might be responsible also for the impaired mitochondrial function in cloned embryos/fetuses

Embryonic Diapause Is Conserved across Mammals

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders

Evaluation of anesthesia and reproductive performance upon diazepam and xylazine injection in rats

The aims of the current study were to evaluate the effects of diazepam (D) and/or xylazine (X) on the induction of sedation and anesthesia, performing ovarian transplantation and litter size upon administration during pregnancy. One hundred and five albino rat females were classified randomly into four groups given intraperitoneal (IP) injection of physiological saline (group I), diazepam (6.2 mg /kg body weight -group II), xylazine (13.2 mg/kg/body weight -group III) and 6.2 mg/kg body weight diazepam and 13.2 mg/kg body weight xylazine (group IV). Induction of sedation and anesthesia were monitored. The onset of anesthesia, duration of surgical anesthesia and sleep time after injections was recorded. Rectal temperatures were monitored at 0, 20, 40, 60 and 120 min after injection. Blood samples were collected at 0 and 120 min post injection for determination of hemoglobin, glucose and urea concentrations. Ovarian transplantation was performed in females anesthesized with DX drugs. Mated females were injected with physiological saline, 6.2 mg/kg diazepam and/or 13.2 mg/kg xylazine at day 4, 11 and 18 of pregnancy and litter sizes were recorded. Sedation was obtained after injection of diazepam or xylazine alone whereas surgical anesthesia was obtained upon injection of both diazepam and xylazine. The onset of anesthesia, duration of anesthesia and sleep time after DX injection was 4.6, 144.1 and 79.1 min, respectively. Rectal temperature decreased (P<0.05) after injection of diazepam and/or xylazine. The hemoglobi

Reproductive biotechnology and critically endangered species: Merging in\ua0vitro gametogenesis with inner cell mass transfer

A fifth of mammalian species face the risk of extinction. A variety of stresses, and lack of sufficient resources and political endorsement, mean thousands of further extinctions in the coming years. Once a species has declined to a mere few individuals, in situ efforts seem insufficient to prevent its extinction. Here we propose a roadmap to overcome some of the current roadblocks and facilitate rejuvenation of such critically endangered species. We suggest combining two advanced assisted reproductive technologies to accomplish this task. The first is the generation of gametes from induced pluripotent stem cells, already demonstrated in mice. The second is to \u2018trick\u2019 the immunological system of abundant species\u2019 surrogate mothers into believing it carries conceptus of its own species. This can be achieved by transferring the inner cell mass (ICM) of the endangered species into a trophoblastic vesicle derived from the foster mother's species. Such synthesis of reproductive biotechnologies, in association with in situ habitat conservation and societal changes, holds the potential to restore diversity and accelerate the production of animals in the most endangered species on Eart

Autophagy and apoptosis: parent-of-origin genome-dependent mechanisms of cellular self-destruction

Functional genomic imprinting is necessary for the transfer of maternal resources to mammalian embryos. Imprint-free embryos are unable to establish a viable placental vascular network necessary for the transfer of resources such as nutrients and oxygen. How the parental origin of inherited genes influences cellular response to resource limitation is currently not well understood. Because such limitations are initially realized by the placenta, we studied how maternal and paternal genomes influence the cellular self-destruction responses of this organ specifically. Here, we show that cellular autophagy is prevalent in androgenetic (i.e. having only a paternal genome) placentae, while apoptosis is prevalent in parthenogenetic (i.e. having only a maternal genome) placentae. Our findings indicate that the parental origin of inherited genes determines the placenta's cellular death pathway: autophagy for androgenotes and apoptosis for parthenogenotes. The difference in time of arrest between androgenotes and parthenogenotes can be attributed, at least in part, to their placentae's selective use of these two cell death pathways. We anticipate our findings to be a starting point for general studies on the parent-of-origin regulation of autophagy. Furthermore, our work opens the door to new studies on the involvement of autophagy in pathologies of pregnancy in which the restricted transfer of maternal resources is diagnosed

Impaired placental vasculogenesis compromises the growth of sheep embryos developed in vitro

To evaluate how assisted reproductive technologies (ART) affect vasculogenesis of the developing conceptus, we analyzed placental and fetal development of in vitro-produced (IVP) sheep embryos. Pregnancies produced by ART carry increased risk of low birth weight, though what causes this risk remains largely unknown. We recently reported that developmental arrest of sheep conceptuses obtained by ART is most pronounced when the cardiovascular system develops (Days 20-30 of development). A total of 86 IVP blastocysts (2-4 per ewe) were surgically transferred to 30 recipient sheep 6 days after estrus; 20 sheep were naturally mated (control). Conceptuses were recovered from sheep at Days 20, 22, 26, and 30 of gestation and morphologically evaluated. Then, the conceptuses and part of their placentae (chorion-allantois) were fixed for histological and immunohistochemical analysis and snap-frozen in liquid nitrogen for subsequent mRNA expression analysis. Results demonstrate that the cardiovascular systems of sheep IVP conceptuses were severely underdeveloped. Pericardial and placental hemorrhages were noted in a majority (5/7) of the dead embryos. In the surviving IVP embryos, the expression of angiogenetic factors was reduced at Day 20. The placental vessels were underdeveloped on Days 20 and 22 (P < 0.05), though placental vasculogenesis was successfully completed on subsequent days. However, low vessel number persisted at Days 26 and 30 (4.6 vs. 5.9 and 6.64 vs. 8.70 per field, respectively; P < 0.05) together with reduced vessel diameter at Day 26 (46.89 vs. 89.92 μm; P < 0.05). In vitro production of sheep embryos induced severely impaired vasculogenesis early in gestation. This may lead to developmental programing problems, such as intrauterine growth restriction of the fetus, resulting in long-term health consequences for the offspring, such as cardiovascular diseases

Experimental design of embryonic diapause (ED) induction in ovine blastocysts by transfer into ovariectomised pseudo-pregnant mice at 2.5 dpc.

<p>Following uterine flushing, diapausing ovine blastocysts were analyzed or transferred to foster ewes at day 6 after oestrus for full term development. The timing indicated in the diagram refers to embryos.</p

Confirmation of ED in ovine embryos.

<p>(<i>a</i>) Proportion of BrdU- and TUNEL-positive cells in diapausing ovine and murine blastocysts flushed from ovariectomised mouse uteri and in controls (<i>b</i>) qRT-PCR analysis of genes involved in ED control. Genes that positively regulate cell proliferation (<i>PCNA</i>) and signaling (<i>HB-EGF</i>) were not expressed in diapausing ovine blastocysts, while the anti-proliferative gene <i>BTG1</i> was significantly over-expressed. <i>IGF2R</i> mRNA expression did not differ statistically between diapausing and control blastocysts. (c) Immunolocalization of CB1 (green) in diapausing (middle panel) and control ovine blastocysts (upper panel). Nuclei (red) were visualized with propidium iodide. CB1 expression is higher in diapausing ovine blastocysts. Lower panel: ovine blastocysts incubated with neutralized anti-CB1 antibody showing no positive signal. For each experiment ≥5 blastocysts were used and it was repeated 3–5 times. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033027#s2" target="_blank">Results</a> are mean ± S.E.M. *** p<0.0001, ** p<0.003, *p<0.03.</p