Preconceptional Resveratrol Supplementation Partially Counteracts Age-Related Reproductive Complications in C57BL/6J Female Mice.

Aging is associated with a drastic decline in fertility/fecundity and with an increased risk of pregnancy complications. Resveratrol (RES), a natural polyphenolic compound, has shown anti-oxidant and anti-inflammatory activities in both human and animal models, thus representing a potential therapeutic and prophylactic anti-aging supplement. Here, we investigated whether preconceptional resveratrol supplementation improved reproductive outcomes in mid-aged (8-month-old) and old (12-month-old) C57BL/6J female mice. Female siblings were cohoused and assigned to either RES or vehicle supplementation to drinking water for 10 consecutive weeks. Subsequently, females were mated with non-supplemented males and their pregnancy outcomes were monitored. RES improved mating success in old, but not in mid-aged females, and prevented the occurrence of delivery complications in the latter. These results indicate that preconceptional RES supplementation could partially improve age-related reproductive complications, but it was not sufficient to restore fecundity in female mice at a very advanced age.

Reproductive biotechnology and critically endangered species: Merging in vitro 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 'trick' the immunological system of abundant species' 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 Earth.

In Memoriam - Prof. Andrzej Krzysztof Tarkowski (1933-2016).

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.

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.

Embryonic diapause in humans: time to consider?

BACKGROUND: When a competent blastocyst stage embryo finds itself in an unreceptive uterus, it delays development. In around one hundred species representing various orders, this delay is known to be reversible, but this phenomenon - termed embryonic diapause (ED) - is not considered a general characteristic of all mammals. PRESENTATION OF THE HYPOTHESIS: Recently, however, we demonstrated that a non-diapausing species, the sheep, is capable of ED, suggesting the hypothesis that this is in fact an ancestral trait common to all mammals, including humans. TESTING THE HYPOTHESIS: In spite of the obvious difficulties in testing this idea, we propose a combination of indirect observations on human fertility patients, and direct study of the embryos of non-human primates. IMPLICATIONS OF THE HYPOTHESIS: Support for our hypothesis would require revision of obstetric interventions routinely performed when a human pregnancy extends beyond the due date.

Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

Influence of embryo transfer on embryo preimplantation development.

OBJECTIVE: To investigate the impact of injection speeds of the transferred load on embryo development. DESIGN: A laboratory model for in vitro simulation of ET was developed to investigate the impact of varying injection speeds of the transferred load on embryo development. SETTING: Academic research institutes of reproduction biotechnology and private centers of reproductive medicine. ANIMAL(S): Mouse hybrid F(1) females (C57bl/10 J × CBA-H; N = 15) aged 2-3 months. INTERVENTION(S): In vitro exposure of mouse embryos with either the fast ET (ejection speed, >1 m/s) or slow ET (ejection speed, <0.1 m/s) and consecutive culture for 36 hours. MAIN OUTCOME MEASURE(S): Development rate, morphology and apoptotic index of embryos. RESULT(S): The development rate was the slowest in embryos exposed to the fast ET. Morphological changes in response to ET were observed only among embryos exposed to the fast ET. The mean apoptotic index was 17.6% in the group exposed to the fast ET, 5.6% in the group exposed to the slow ET, and 2.58% in the control group. CONCLUSION(S): A reduction of the ejection speed of the transferred load allows avoidance of a developmental delay and diminishes injury of the embryos. Therefore, it is reasonable to suggest transferring the embryos at the lowest possible ejection speed.

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.

Fluid dynamics during embryo transfer.

OBJECTIVE: To study fluid dynamics during ET. DESIGN: Computational fluid dynamics were applied to calculate fluid velocity changes, dynamic pressure differences, and shear stress in the transferred load for the following injection speeds: 0.1, 1, 6, 12, and 20 m/sec. SETTING: Academic research institute of mechanical engineering and reproduction biotechnology and private centers of reproductive medicine. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fluid velocity, dynamic pressure, and shear stress during injection of the transferred load. RESULT(S): An increase of injection speed for the transferred load increased the shear stress, dynamic pressure, and velocity differences acting on the embryo. The narrowing of the catheter lumen diameter by 20% amplified the transferred fluid velocity by 78%. An embryo positioned in proximity to the catheter's wall was exposed to considerably higher shear stress, dynamic pressure, and velocity difference than an embryo in the center of the catheter's lumen. CONCLUSION(S): The transfer of an embryo should be conducted gently and with minimal injection speed. Any narrowing of the catheter lumen should be eliminated. Preferably the embryo should be kept far from the catheter's wall during injection of the transferred load.

Pressure induced nucleus DNA fragmentation.

PURPOSE: The present study was designed to investigate the impact of pressure on nuclear DNA integrity in viable cells of mouse blastocysts. METHODS: The blastocysts of hybrid F1 females [(C57Bl/10 J × CBA-H);N = 15] aged 2-3 months were exposed into the pressure impulse lasting ~0.021 s and characterized by a positive pressure peak of ~76 mmHg. The nuclear DNA fragmentation index of mouse blastocysts was assessed by TUNEL assay within 60 s after exposure to pressure impulse. RESULTS: The mean nuclear DNA fragmentation index was significantly higher in the experimental group (83%) than in the control group (19.7%); p < 0.001. CONCLUSION(S): A low magnitude pressure impulse can induce nuclear DNA fragmentation in mouse blastocysts. The compression and decompression forces appearing during pressure fluctuations are responsible for the observed DNA shearing.

Influence of embryo transfer on blastocyst viability.

OBJECTIVE: To investigate the impact of injection speeds of the transferred load on embryo viability. DESIGN: Laboratory model for in vitro simulation of embryo transfer (ET). SETTING: Academic research institutes of reproduction biotechnology and private centers of reproductive medicine. ANIMAL(S): Mouse hybrid F1 females, C57bl/10J × CBA-H (N = 15), aged 2 to 3 months. INTERVENTION(S): In vitro exposure of mouse blastocysts to either fast ET with an ejection speed of the transferred load of >1 m/s or slow ET with an ejection speed of <0.1 m/s. MAIN OUTCOME MEASURE(S): Morphologic changes and apoptotic index of blastocysts. RESULT(S): Morphologic changes in response to ET were most prevalent in blastocysts exposed to fast ET. The mean apoptotic index was 52% in the group exposed to fast ET, 25% in the group exposed to slow ET, and 12.8% in control group. CONCLUSION(S): Fast ejection of the transferred load can trigger both morphologic changes and apoptosis in mouse blastocysts. A reduction of the ejection speed of the transferred load minimizes injury to the embryos. Therefore, embryos should be transferred at the lowest possible speed.

Enucleated GV oocytes as recipients of embryonic nuclei in the G1, S, or G2 stages of the cell cycle.

Universal recipients in the G2 phase of mitotic cell cycle (preactivated oocytes, zygotes, blastomeres) accept embryonic nuclei in all the stages of their cell cycle. To test if recipients in the G2 of meiotic cycle (immature oocytes) are universal recipients, mouse germinal vesicle (GV) oocytes were enucleated and reconstructed with blastomere nuclei in the G1, S, or G2 stages. Analysis of their maturation has shown that about 30% of the G1 nuclei and 60% of G2 nuclei allow for normal metaphase II (MII), both in the oocytes with and without the first polar body (1st PB). Among oocytes reconstructed with the S phase nuclei, only 8% or less have normal MII, although 75% of them extrude 1st PB. No phase of donor cell cycle prevented the abnormal acceleration of 1st PB extrusion, found in reconstructed GV oocytes. In conclusion, enucleated GV oocytes are not universal recipients of embryonic nuclei, because they do not accept the S donors. However, both the G1 and G2 donor nuclei can be reprogrammed in the GV oocyte cytoplasm.

Experimental embryonic-somatic chimaerism in the sheep confirmed by random amplified polymorphic DNA assay.

Developmental potencies of sheep somatic cells (foetal fibroblasts, FFs) in chimaeric animals were analysed. FFs from pigmented Polish Heatherhead (wrzosowka) breed were microsurgically injected into morulae or blastocysts of white Polish Merino breed (5 cells to each embryo). In one experiment the cells were stained with vital fluorescent dye PKH26, and chimaeric blastocysts were cultured in vitro to confirm the presence of fluorescent cells. In the majority of experiments the blastocysts were transferred to synchronized recipient ewes for development until term. Cultured embryonic cells (CEC), earlier known to produce chimaeras, were injected into blastocysts in control experiments. Seven young were born from FF-injected embryos and three were born from CEC-injected ones. All of them were white, but all three control lambs and three experimental lambs showed small areas of skin pigmentation, which indicated Heatherhead CEC or FF contribution. Tissue samples originating from three germ layers were taken from two FFs-originating presumably chimaeric lambs (male and female) at the age of one month for DNA analysis. The random amplified polymorphic DNA-PCR method supplied two markers of chimaerism, which were amplification products of 643 bp and 615 bp long DNA fragments, found in tissues of experimental lambs as well as in FFs, but not in the blood of parents of blastocysts. The 643 bp marker was found in the majority of tissues of both lambs. The 615 bp amplicon was detected in the skin and lungs of the female lamb and in the hooves of the male lamb. Our data show that foetal fibroblasts introduced to sheep blastocysts can participate in development and can contribute to all tissue lineages up to at least one month of age.

Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration.

Melatonin promotes mouse embryo development in vitro. An effect of melatonin on bovine embryo development is described here. Slaughterhouse derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes were cultured for 2 days in CR1aaLA medium supplemented with melatonin (10(-4) m) or without melatonin (control). Culture was performed under two different gas atmospheres containing physiological (7%) or atmospheric (20%) oxygen concentrations (2x2 factorial analysis). After day 2, embryos from each treatment group developed to at least four-cell stage, were cultured without melatonin until day 10 at optimum 7% O2 atmosphere. Blastocyst formation rates of presumptive zygotes and of four-cell embryos were calculated for each group. Significant interactions between oxygen tension and the melatonin treatment were found. Out of four-cell embryos put into in vitro culture after initial incubation in medium containing melatonin, decreased blastocyst rate was observed in melatonin group (47.7%) compared with control (67.7%; P=0.0327) when lower oxygen concentration was applied. A beneficial effect of melatonin was observed in 20% O2: out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in melatonin versus 32 of 63 (50.8%; P=0.0458) blastocysts that developed in control group. In conclusion, beneficial or harmful effects of melatonin on bovine embryo development in vitro were observed, depending on the oxygen tension during the treatment.

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.

Mouse zygotes as recipients in embryo cloning.

Zygotes have not been recognized as nuclear recipients since enucleated zygotes receiving nuclei from beyond two-cell stage embryos are not able to form blastocysts. In the present study, a new technique of zygote enucleation is presented, which consists in selectively removing the nuclear membrane with genetic material of pronuclei, but leaving other pronuclear components in the cytoplasm. With selective enucleation it is possible - after transfer of eight-cell stage nuclei - to obtain 70.5 and 7.8% of preimplantation and full-term development respectively. Origin of cloned mice from introduced nuclei was confirmed by the coat colour and glucose phosphate isomerase (GPI) isozyme of the donor. We suggest that some pronuclear factors - taken away from the zygotes in the karyoplasts upon classical enucleation - are needed to reprogram the introduced nuclei.

Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology.

The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds were chimeras. The effect of donor cells on the reproduction and physiology of the recipients was evident.

Meiotic maternal chromosomes introduced to the late mouse zygote are recruited to later embryonic divisions.

The aim of this study was to investigate the fate of an additional female genome introduced to a dividing zygote. Maternal chromatin in the form of karyoplasts containing a metaphase II spindle were fused to zygotes blocked in anaphase or telophase of the first cleavage. Permanent preparations made 20-40 min after fusion at anaphase revealed that the donor maternal chromosomes had entered anaphase or telophase in 16 out of 18 cases. A further two groups of embryos that were fused at either anaphase or anaphase/telophase were cultured to the first division. Division occurred 50 min after fusion in both groups of embryos (86 and 85.1%, respectively), of which most divided to two cells (80 and 71.6% of total) and the remainder divided to three cells. About two thirds of two-cell embryos contained an extra nucleus in one blastomere. Nuclei containing donor maternal chromosomes reached a similar size to recipient nuclei in 68% of embryos derived from anaphase-blocked zygotes, in contrast to 31.1% of embryos derived from anaphase/telophase-blocked embryos. Replication of DNA in donor nuclei closely followed the timing and intensity of that in control embryos. When fixed 24 hr after fusion, one third of embryos were still at the two-cell stage, with one or both blastomeres showing a single metaphase plate of the second cleavage. In the remaining embryos, three or four cells were present, some containing two nuclei. Blastocysts developed in 50% of fused embryos and three young were born after transfer of cleaving hybrid embryos to recipients. Chromosome preparations from bone marrow of the young contained 3-4 tetraploid metaphase plates per several hundred plates counted compared with none in control embryos. In conclusion, additional maternal chromosomes can be introduced at the late-dividing zygote and join the embryonic cell cycles during subsequent divisions. This method may provide a useful approach for studying changes specific to the maternal genome during early cell cycles of the mammalian embryo.

Reconstruction of enucleated mouse germinal vesicle oocytes with blastomere nuclei.

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4- or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About one-third of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.