Long-run real-time PCR analysis of repetitive nuclear elements as a novel tool for DNA damage quantification in single cells: an approach validated on mouse oocytes and fibroblasts.

Since DNA damage is of great importance in various biological processes, its rate is frequently assessed both in research studies and in medical diagnostics. The most precise methods of quantifying DNA damage are based on real-time PCR. However, in the conventional version, they require a large amount of genetic material and therefore their usefulness is limited to multicellular samples. Here, we present a novel approach to long-run real-time PCR-based DNA-damage quantification (L1-LORD-Q), which consists in amplification of long interspersed nuclear elements (L1) and allows for analysis of single-cell genomes. The L1-LORD-Q was compared with alternative methods of measuring DNA breaks (Bioanalyzer system, γ-H2AX foci staining), which confirmed its accuracy. Furthermore, it was demonstrated that the L1-LORD-Q is sensitive enough to distinguish between different levels of UV-induced DNA damage. The method was validated on mouse oocytes and fibroblasts, but the general idea is universal and can be applied to various types of cells and species.

Persistent organic pollutants affect steroidogenic and apoptotic activities in granulosa cells and reactive oxygen species concentrations in oocytes in the mouse.

CONTEXT: The destruction of granulosa cells (GCs), the main functional cell type in the ovary, prevents steroid hormone production, which in turn may damage oocytes, resulting in ovarian failure. The accumulation of a number of persistent organic pollutants (POPs) in the ovarian follicular fluid (FF) has been documented, which raises serious questions regarding their impact on female fertility. AIMS: We aimed to determine whether a mixture of POPs reflecting the profile found in FF influences mouse GCs or oocyte function and viability. METHODS: A mixture of POPs, comprising perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene, was used. In addition to using the exact concentration of POPs previously measured in human FF, we tested two other mixtures, one with10-fold lower and another with 10-fold higher concentrations of each POP. KEY RESULTS: Steroidogenesis was disrupted in GCs by the POP mixture, as demonstrated by lower oestradiol and progesterone secretion and greater lipid droplet accumulation. Furthermore, the POP mixture reduced GC viability and increased apoptosis, assessed using caspase-3 activity. The POP mixture significantly increased the number of oocytes that successfully progressed to the second meiotic metaphase and the oocyte reactive oxygen species (ROS) concentration. CONCLUSIONS: Thus, a mixture of POPs that are typically present in human FF has detrimental effects on ovarian function: it reduces the viability of GCs, and increases the oocyte concentrations of ROS. IMPLICATIONS: These results indicate that chronic exposure to POPs adversely affects female reproductive health.

Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

Cellular mechanisms ensuring the mammalian female germ cells quality / Komórkowe mechanizmy zapewniajace jakosc zenskich komórek rozrodczych ssaków.

Gametes are extremely differentiated cells participating in the fertilization to give the beginning of a new life. Except enabling fertilization, however, the fully functional gamete, should also guarantee full and undisturbed development of the whole individual. The aim of this article is to approximate the mechanisms which occur during mammalian oogenesis which are crucial for ensuring the proper course of development as well as the quality of the genetic material transmitted to the progeny.

Mouse single oocyte imaging by MALDI-TOF MS for lipidomics.

Reproductive cells are a very special kind of material for the analysis. Depending on the species, their dimensions allow for the application of mass spectrometry imaging-based techniques to receive a reasonable data for interpretation of their condition without any additional sample preparation steps, except for typical sample preparation characteristic for IMS protocols. A comparison between lipid profiles of oocytes could answer the question of the overall quality of the cells in the function of time or conditions of storage. Even tiny differences in the lipid profiles, but still detectable by bioinformatic analysis, could be crucial for the estimation of the conditions of the cells in various stages of development or aging. In our study, MALDI-TOF/TOF MSI was used to analyze and visualize the single oocytes. We deposited the cells on the transparent indium-tin-oxide (ITO) glass and marked their positions, which allowed for the fast localization of the cells and precise laser targeting in the ion source. We also optimized the usage of different MALDI matrices and different approaches. The proposed way of measurement allows analyzing quite a significant quantity of oocytes in a reasonably short time. During the analysis, the lipid composition of the single cell was successfully estimated in a conventional usage of the MALDI ion source, and the localization of lipids was confirmed by imaging mass spectrometry (IMS) analysis. The observed quantity of the lipids allowed for the application of the LIFT™ technique to obtain MS/MS spectra sufficient for lipids' unambiguous identification. We hope that our idea of the oocyte analysis will help to elucidate chemical changes that accompany different processes in which oocytes are involved. There could be such fascinating phenomena as the oocyte maturation, changes in the lipid components during their storage, and much more.

Aging deteriorates quality of sperm produced by male mice with partial Yq deletion.

The aim of the study was to assess the cumulative effects of aging and Y-chromosome long arm deletion on sperm quality parameters. Motility, mitochondrial activity, and head morphology were evaluated for sperm of 3- and 12-month-old males from B10.BR-Ydel and B10.BR congenic mouse strains. The study revealed that quality and fertilizing potential of sperm produced by younger and older B10.BR males persist on similar levels, but worsen significantly with age of B10.BR-Ydel males. The findings imply that partial Yq deletions might be more harmful for spermiogenesis in advancing age and may be applicable to other species including humans. ABBREVIATIONS: AZF: azoospermia factor; MSYq: male-specific region of the Y-chromosome long arm.

Mitochondrial functionality in female reproduction.

In most animal species female germ cells are the source of mitochondrial genome for the whole body of individuals. As a source of mitochondrial DNA for future generations the mitochondria in the female germ line undergo dynamic quantitative and qualitative changes. In addition to maintaining the intact template of mitochondrial genome from one generation to another, mitochondrial role in oocytes is much more complex and pleiotropic. The quality of mitochondria determines the ability of meiotic divisions, fertilization ability, and activation after fertilization or sustaining development of a new embryo. The presence of normal number of functional mitochondria is also crucial for proper implantation and pregnancy maintaining. This article addresses issues of mitochondrial role and function in mammalian oocyte and presents new approaches in studies of mitochondrial function in female germ cells.

Prediction of Developmentally Competent Chromatin Conformation in Mouse Antral Oocytes.

Mouse prophase oocytes isolated from antral follicles may possess two alternative types of chromatin configuration: NSN configuration represents more dispersed chromatin and is characteristic mainly for growing oocytes whereas SN configuration, attained upon oocyte growth, comprises more condensed chromatin with a significant fraction concentrated around the nucleolus. Importantly, fully grown oocytes isolated from antral follicles represent a non-homogenous population in which some oocytes posses NSN-type and others SN-type of chromatin conformation. From these two, only oocytes with SN configuration are able to complete full development upon fertilization. We show that among mouse oocytes isolated from antral follicles, those surrounded by cumulus cells were larger and more frequently possessed SN chromatin than oocytes lacking the complete cumulus cell layer. Females primed with PMSG gave a higher number of oocytes with a complete layer of cumulus cells and the frequency of oocytes with SN chromatin was also elevated. Within the whole population of isolated antral oocytes, we observed subtle variation in size which allowed fractionation of oocytes under a stereomicroscope into groups representing oocytes of slightly different sizes. The occurrence of SN chromatin configuration was highly dependent on the oocyte size and its frequency increased gradually in subsequent size groups reaching 95-100% in the group representing the largest oocytes. These findings demonstrate that the subtle differences in the size of antral oocytes allow prediction of the status of their chromatin, thus providing a simple, fast, non-invasive and non-expensive way to select good quality oocytes for ART purposes in mammals.

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

Therapeutic potential of somatic cell nuclear transfer for degenerative disease caused by mitochondrial DNA mutations.

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal.

Spindle assembly checkpoint regulation of chromosome segregation in mammalian oocytes.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that monitors the quality of the spindle during division and blocks anaphase entry in the presence of anomalies that could result in erroneous segregation of the chromosomes. Because human aneuploidy is mainly linked to the erroneous segregation of genetic material in oocytes, the issue of the effectiveness of the SAC in female meiosis is especially important. The present review summarises our understanding of the SAC control of mammalian oocyte meiosis, including its possible impact on the incidence of embryonic aneuploidy. Owing to the peculiarities of cell cycle control in female meiosis, the integration of the SAC within such a specific environment results in several unusual situations, which are also discussed.

Free-hand bisection of mouse oocytes and embryos.

Mouse oocytes and zygotes are semitransparent and large cells approximately 80 µm in diameter. Bisection is one of the easiest ways for performing micromanipulations on such cells. It allows living sister halves or smaller fragments to be obtained, which can be cultured and observed for long periods of time. Bisection can be used for different kinds of experiments such as analysis of nucleo-cytoplasmic interactions, the relationship between different cellular structures or between different parts of embryos, eventually for analyzing the developmental potential of embryonic fragments. Oocyte or embryo halves can be examined by immunostaining, by measuring different cellular functions and by Western blot and genetic analysis (e.g., RT-PCR). Here we describe a detailed protocol for the free-hand bisection of mouse zona pellucida-free oocytes and embryos on an agar layer using a glass needle.

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

Spindle assembly checkpoint-related meiotic defect in oocytes from LT/Sv mice has cytoplasmic origin and diminishes in older females.

The spindle assembly checkpoint (SAC) ensures proper segregation of chromosomes by delaying anaphase onset until all kinetochores are properly attached to the spindle microtubules. Oocytes from the mouse strain LT/Sv arrest at the first meiotic metaphase (MI) due to, as reported recently, enormously prolonged activity of the SAC. We compared the dynamics of cyclin B1-GFP degradation, the process which is a measure of the SAC activity, in chromosomal and achromosomal halves of LT/Sv oocytes. In chromosome-containing oocyte halves arrested at MI, cyclin B1-GFP was not degraded indicating active SAC. However, in the halves lacking chromosomes, which is a condition precluding the SAC function, degradation always occurred confirming that MI arrest in LT/Sv oocytes is SAC dependent. Transferring the germinal vesicle (GV) from LT/Sv oocytes into the enucleated oocytes from wild-type mice resulted in the progression through meiosis one, indicating that a SAC-activating defect in LT/Sv oocytes is cytoplasmic, yet can be rescued by foreign cytoplasm. These results may help to define the etiology of the human infertility related to the oocyte MI arrest, indicating the involvement of the SAC as likely candidate, and point to GV transfer as the possible therapy. Finally, we found that majority of oocytes isolated from old LT/Sv mice complete the first meiosis. Reciprocal transfers of the GV between the oocytes from young and old LT/Sv females suggest that the factor(s) responsible for the reversal of the phenotype in oocytes from old mice is located both in the GV and in the cytoplasm.

Association of TCTP with centrosome and microtubules.

Translationally Controlled Tumour Protein (TCTP) associates with microtubules (MT), however, the details of this association are unknown. Here we analyze the relationship of TCTP with MTs and centrosomes in Xenopus laevis and mammalian cells using immunofluorescence, tagged TCTP expression and immunoelectron microscopy. We show that TCTP associates both with MTs and centrosomes at spindle poles when detected by species-specific antibodies and by Myc-XlTCTP expression in Xenopus and mammalian cells. However, when the antibodies against XlTCTP were used in mammalian cells, TCTP was detected exclusively in the centrosomes. These results suggest that a distinct pool of TCTP may be specific for, and associate with, the centrosomes. Double labelling for TCTP and γ-tubulin with immuno-gold electron microscopy in Xenopus laevis oogonia shows localization of TCTP at the periphery of the γ-tubulin-containing pericentriolar material (PCM) enveloping the centriole. TCTP localizes in the close vicinity of, but not directly on the MTs in Xenopus ovary suggesting that this association requires unidentified linker proteins. Thus, we show for the first time: (1) the association of TCTP with centrosomes, (2) peripheral localization of TCTP in relation to the centriole and the γ-tubulin-containing PCM within the centrosome, and (3) the indirect association of TCTP with MTs.

Restricted myogenic potential of mesenchymal stromal cells isolated from umbilical cord.

Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Wharton's jelly have been shown to be able to differentiate into various cell types. Thus, as they are readily available and do not raise any ethical issues, these cells are considered to be a potential source of material that can be used in regenerative medicine. In our previous study, we tested the potential of whole mononucleated fraction of human umbilical cord blood cells and showed that they are able to participate in the regeneration of injured mouse skeletal muscle. In the current study, we focused at the umbilical cord mesenchymal stromal cells isolated from Wharton's jelly. We documented that limited fraction of these cells express markers of pluripotent and myogenic cells. Moreover, they are able to undergo myogenic differentiation in vitro, as proved by coculture with C2C12 myoblasts. They also colonize injured skeletal muscle and, with low frequency, participate in the formation of new muscle fibers. Pretreatment of Wharton's jelly mesenchymal stromal cells with SDF-1 has no impact on their incorporation into regenerating muscle fibers but significantly increased muscle mass. As a result, transplantation of mesenchymal stromal cells enhances the skeletal muscle regeneration.

DNA methylation, histone modifications and behaviour of AKAP95 during mouse oocyte growth and upon nuclear transfer of foreign chromatin into fully grown prophase oocytes.

The poor efficiency of mammalian cloning is due to inappropriate/incomplete epigenetic reprogramming of the donor chromatin. As the success in reprogramming of the donor nucleus may require activity of similar mechanisms which reprogram the chromatin in the course of gametogenesis, we decided to follow the status of some epigenetic markers in the late phase of oogenesis in mice, i.e. in prophase oocytes during their growth and after completion of the growth phase. Our analysis reveals an increase in the level of global DNA methylation starting in oocytes with diameters around 60 microm which was further elevated until completion of oocyte growth. A similar increase was observed in respect to the acetylation of histone H4. On the other hand, the methylation of histone H4 Arg3 was constantly high until the end of oocyte growth, although it differed between fully grown oocytes depending on the type of spatial chromatin organization. We have also studied the AKAP95 protein which was abundant at earlier stages but decreased in fully grown oocytes according to changes in their chromatin organization. The nuclear transfer of different types of donor nuclei with hypomethylated DNA into fully grown prophase oocytes did not increase the global level of methylation of transferred foreign chromatin, regardless if the recipient oocyte was devoid of its own nucleus or its nucleus was left intact. This suggests a major problem in the ability of recipient oocytes to modify donor DNA methylation.

A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I.

The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size.

Spindle assembly checkpoint-related failure perturbs early embryonic divisions and reduces reproductive performance of LT/Sv mice.

The phenotype of the LT/Sv strain of mice is manifested by abnormalities in oocyte meiotic cell-cycle, spontaneous parthenogenetic activation, teratomas formation, and frequent occurrence of embryonic triploidy. These abnormalities lead to the low rate of reproductive success. Recently, metaphase I arrest of LT/Sv oocytes has been attributed to the inability to timely inactivate the spindle assembly checkpoint (SAC). As differences in meiotic and mitotic SAC functioning were described, it remains obscure whether this abnormality is limited to the meiosis or also impinges on the mitotic divisions of LT/Sv embryos. Here, we show that a failure to inactivate SAC affects mitoses during preimplantation development of LT/Sv embryos. This is manifested by the prolonged localization of MAD2L1 on kinetochores of mitotic chromosomes and abnormally lengthened early embryonic M-phases. Moreover, LT/Sv embryos exhibit elevated frequency of abnormal chromosome separation during the first mitotic division. These abnormalities participate in severe impairment of preimplantation development and significantly decrease the reproductive success of this strain of mice. Thus, the common meiosis and mitosis SAC-related failure participates in a complex LT/Sv phenotype.

Metaphase I arrest in LT/Sv mouse oocytes involves the spindle assembly checkpoint.

During meiotic maturation, the majority of oocytes from LT/Sv mice arrest at metaphase I. However, anaphase may be induced through parthenogenetic activation. If this happens within the ovary, it often results in the development of ovarian teratomas. Here, we show that the induction of first meiotic anaphase in LT/Sv oocytes results in incorrect chromosome segregation. In search of the molecular basis of this complex phenotype, we analyzed the localization/destruction of cohesins, as well as the function of the components of the spindle assembly checkpoint (SAC). Both localization and removal of meiotic cohesin REC8 from chromosomes are unperturbed. In contrast, there is prolonged localization of SAC proteins BUB1 and MAD2L1 (MAD2) at the metaphase I kinetochores in mutant oocytes compared with the wild-type. Interfering with BUB1 function through expression of a dominant-negative mutant protein resulted in the increase of the number of LT/Sv oocytes completing the first meiosis, which indicates SAC involvement in metaphase I arrest. These data show for the first time that there is a direct link between the SAC function and the heritable meiotic incompetence of a mammalian oocyte.