Oocyte activation is a cytoplasm-confined event so far: what about the nucleus?

The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.

New approaches for long-term conservation of rooster spermatozoa.

In contrast to the livestock industry, sperm cryopreservation has not yet been successfully established in the poultry industry. This is because poultry sperm cells have a unique shape and membrane fluidity, differing from those of livestock sperm. The objective of this review is to discuss the cellular and molecular characteristics of rooster spermatozoa as a cause for their generally low freezability. Furthermore, here, we discuss novel developments in the field of semen extenders, cryoprotectants, and freezing processes, all with the purpose of increasing the potential of rooster sperm cryopreservation. Currently, it is very important to improve cryopreservation of rooster sperm on a global scale for the protection of gene resources due to the incidence of epidemics such as avian influenza.

Omne vivum ex ovo: the oocyte reprogramming and remodeling activities.

In brief: Understanding the establishment of post-fertilization totipotency has broad implications for modern biotechnologies. This review summarizes the current knowledge of putative egg components governing this process following natural fertilization and after somatic cell nuclear transfer. Abstract: The mammalian oocyte is a unique cell, and comprehending its physiology and biology is essential for understanding fertilization, totipotency and early events of embryogenesis. Consequently, research in these areas influences the outcomes of various technologies, for example, the production and conservation of laboratory and large animals with rare and valuable genotypes, the rescue of the species near extinction, as well as success in human assisted reproduction. Nevertheless, even the most advanced and sophisticated reproductive technologies of today do not always guarantee a favorable outcome. Elucidating the interactions of oocyte components with its natural partner cell - the sperm or an 'unnatural' somatic nucleus, when the somatic cell nucleus transfer is used is essential for understanding how totipotency is established and thus defining the requirements for normal development. One of the crucial aspects is the stoichiometry of different reprogramming and remodeling factors present in the oocyte and their balance. Here, we discuss how these factors, in combination, may lead to the formation of a new organism. We focus on the laboratory mouse and its genetic models, as this species has been instrumental in shaping our understanding of early post-fertilization events.

Interspecific ICSI for the Assessment of Sperm DNA Damage: Technology Report.

Xenogenic mammalian sperm heads injected into mouse ovulated oocytes decondense and form pronuclei in which sperm DNA parameters can be evaluated. We suggest that this approach can be used for the assessment of sperm DNA damage level and the evaluation of how certain sperm treatments (freezing, lyophilization, etc.) influence the quality of spermatozoa.

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Scientific and technological approaches to improve SCNT efficiency in farm animals and pets.

The birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here, we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development.

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

Function of atypical mammalian oocyte/zygote nucleoli and its implications for reproductive biology and medicine.

Mammalian oocytes/zygotes contain atypical nucleoli that are composed exclusively of a dense fibrillar material. It has been commonly accepted that these nucleoli serve as a repository of components that are used later on, as the embryo develops, for the construction of typical tripartite nucleoli. Indeed, when nucleoli were removed from immature oocytes (enucleolation) and these oocytes were then matured, fertilized or parthenogenetically activated, development of the produced embryos ceased after one or two cleavages with no detectable nucleoli in nuclei. This indicated that zygotic nucleoli originate exclusively from oocytes, i.e. are maternally inherited. Recently published results, however, do not support this developmental biology dogma and demonstrate that maternal nucleoli in one-cell stage embryos are necessary only during a very short time period after fertilization when they serve as a major heterochromatin organizing structures. Nevertheless, it still remains to be determined, which other functions/roles the atypical oocyte/zygote nucleoli eventually have.

Enucleolation and nucleolus transfer in mammalian oocytes and zygotes.

The oocyte GV/GVs (germinal vesicle/germinal vesicles) and zygot PN/PNs (pronucleus/pronuclei) of some mammals contain clearly visible nucleoli which exhibit an atypical morphological structure. These nucleoli (NCLs) can be relatively easily manipulated, i.e. removed from GVs/PNs or eventually transferred into another oocyte/zygote. Thus, with the help of micromanipulation techniques it was possible to uncover the real function(s) they play in processes of oocyte maturation and early embryonic development. The purpose of our review is to describe briefly the micromanipulation techniques that can be used for oocyte/zygote nucleoli manipulation. Moreover, we present some examples of results that were obtained in nucleolus manipulation experiments.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

Can Nucleoli Be Markers of Developmental Potential in Human Zygotes?

In 1999, Tesarik and Greco reported that they could predict the developmental potential of human zygotes from a single static evaluation of their pronuclei. This was based on the distribution and number of specific nuclear organelles - the nucleoli. Recent studies in mice show that nucleoli play a key role in parental genome restructuring after fertilization, and that interfering with this process may lead to developmental failure. These studies thus support the Tesarik-Greco evaluation as a potentially useful method for selecting high-quality embryos in human assisted reproductive technologies. In this opinion article we discuss recent evidence linking nucleoli to parental genome reprogramming, and ask whether nucleoli can mirror or be used as representative markers of embryonic parameters such as chromosome content or DNA fragmentation.

De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes.

The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials.

Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.

Pluripotent stem cells from maturing oocytes.

Embryonic stem cells are mostly derived from mature oocytes that were either fertilized or activated parthenogenetically and then reached the blastocyst stage. From the cell cycle perspective, fertilization or activation induces the exit from meiosis, decondensation of oocyte chromosomes, and the entry into mitosis. Decondensation of oocyte chromatin with subsequent formation of nuclei can be, however, induced at any postgerminal vesicle breakdown meiotic maturation stage. In this article, we discuss the possibility of cleavage of transformed maturing oocytes and whether they can reach the blastocyst stage, from which pluripotent stem cell lines could be derived.

The ups and downs of somatic cell nucleus transfer (SCNT) in humans.

Achieving successful somatic cell nuclear transfer (SCNT) in the human and subhuman primate relative to other mammals has been questioned for a variety of technical and logistical issues. Here we summarize the gradual evolution of SCNT technology from the perspective of oocyte quality and cell cycle status that has recently led to the demonstration of feasibility in the human for deriving chromosomally normal stem cells lines. With these advances in hand, prospects for therapeutic cloning must be entertained in a conscientious, rigorous, and timely fashion before broad spectrum clinical applications are undertaken.

RNA polymerase II transcriptional silencing in growing and fully grown germinal vesicle oocytes isolated from gonadotropin-stimulated and non-stimulated gilts.

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.

Production of giant mouse oocyte nucleoli and assessment of their protein content.

Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.

Genome of non-living cells: trash or recycle?

Reproductive technologies have been often used as a tool in research not strictly connected with developmental biology. In this study, we retrace the experimental routes that have led to the adoption of two reproductive technologies, ICSI and somatic cell nuclear transfer (SCNT), as biological assays to probe the 'functionality' of the genome from dead cells. The structural peculiarities of the spermatozoa nucleus, namely its lower water content and its compact chromatin structure, have made it the preferred cell for these experiments. The studies, primarily focused on mice, have demonstrated an unexpected stability of the spermatozoa nuclei, which retained the capacity to form pronuclei once injected into the oocytes even after severe denaturing agents like acid treatment and high-temperature exposure. These findings inspired further research culminating in the production of mice after ICSI of lyophilized spermatozoa. The demonstrated non-equivalence between cell vitality and nuclear vitality in spermatozoa prompted analogous studies on somatic cells. Somatic cells were treated with the same physical stress applied to spermatozoa and were injected into enucleated sheep oocytes. Despite the presumptive fragile nuclear structure, nuclei from non-viable cells (heat treated) directed early and post-implantation embryonic development on nuclear transfer, resulting in normal offspring. Recently, lyophilized somatic cells used for nuclear transfer have developed into normal embryos. In summary, ICSI and SCNT have been useful tools to prove that alternative strategies for storing banks of non-viable cells are realistic. Finally, the potential application of freeze-dried spermatozoa and cells is also discussed.

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig.

In mammals, the nucleolus of full-grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full-grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non-treated or actinomycin D-treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re-injection of nucleoli from growing oocytes (23%), but not when nucleoli from full-grown oocytes were re-injected into enucleolated, growing oocytes (49%). When enucleolated, full-grown oocytes were injected with nucleoli from growing or full-grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full-grown oocytes injected with nucleoli from full-grown oocytes matured to metaphase II (56%), whereas injection with growing-oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing-oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full-grown oocyte nucleolus has lost the ability.