The timing of pronuclear transfer critically affects the developmental competence and quality of embryos.

Pronuclear transfer has been successfully used in human-assisted reproduction to suppress the adverse effects of a defective oocyte cytoplasm or to bypass an idiopathic developmental arrest. However, the effects of the initial parental genome remodelling in a defective cytoplasm on the subsequent development after pronucleus transfer have not been systematically studied. By performing pronuclear transfer in pre-replication and post-replication mouse embryos, we show that the timing of the procedure plays a critical role. Although apparently morphologically normal blastocysts were obtained in both pre- and post-replication pronuclear transfer groups, post-replication pronuclear transfer led to a decrease in developmental competence and profound changes in embryonic gene expression. By inhibiting the replication in the abnormal cytoplasm before pronuclear transfer into a healthy cytoplasm, the developmental potential of embryos could be largely restored. This shows that the conditions under which the first embryonic replication occurs strongly influence developmental potential. Although pronuclear transfer is the method of choice for mitigating the impact of a faulty oocyte cytoplasm on early development, our results show that the timing of this intervention should be restricted to the pre-replication phase.

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.

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.

Acrosin is essential for sperm penetration through the zona pellucida in hamsters.

During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.

De novo emergence, existence, and demise of a protein-coding gene in murids.

BACKGROUND: Genes, principal units of genetic information, vary in complexity and evolutionary history. Less-complex genes (e.g., long non-coding RNA (lncRNA) expressing genes) readily emerge de novo from non-genic sequences and have high evolutionary turnover. Genesis of a gene may be facilitated by adoption of functional genic sequences from retrotransposon insertions. However, protein-coding sequences in extant genomes rarely lack any connection to an ancestral protein-coding sequence. RESULTS: We describe remarkable evolution of the murine gene D6Ertd527e and its orthologs in the rodent Muroidea superfamily. The D6Ertd527e emerged in a common ancestor of mice and hamsters most likely as a lncRNA-expressing gene. A major contributing factor was a long terminal repeat (LTR) retrotransposon insertion carrying an oocyte-specific promoter and a 5' terminal exon of the gene. The gene survived as an oocyte-specific lncRNA in several extant rodents while in some others the gene or its expression were lost. In the ancestral lineage of Mus musculus, the gene acquired protein-coding capacity where the bulk of the coding sequence formed through CAG (AGC) trinucleotide repeat expansion and duplications. These events generated a cytoplasmic serine-rich maternal protein. Knock-out of D6Ertd527e in mice has a small but detectable effect on fertility and the maternal transcriptome. CONCLUSIONS: While this evolving gene is not showing a clear function in laboratory mice, its documented evolutionary history in Muroidea during the last ~ 40 million years provides a textbook example of how a several common mutation events can support de novo gene formation, evolution of protein-coding capacity, as well as gene's demise.

Nucleus reprogramming/remodeling through selective enucleation (SE) of immature oocytes and zygotes: a nucleolus point of view.

It is now approximately 25 years since the sheep Dolly, the first cloned mammal where the somatic cell nucleus from an adult donor was used for transfer, was born. So far, somatic cell nucleus transfer, where G1-phase nuclei are transferred into cytoplasts obtained by enucleation of mature metaphase II (MII) oocytes followed by the activation of the reconstructed cells, is the most efficient approach to reprogram/remodel the differentiated nucleus. In general, in an enucleated oocyte (cytoplast), the nuclear envelope (NE, membrane) of an injected somatic cell nucleus breaks down and chromosomes condense. This condensation phase is followed, after subsequent activation, by chromatin decondensation and formation of a pseudo-pronucleus (i) whose morphology should resemble the natural postfertilization pronuclei (PNs). Thus, the volume of the transferred nuclei increases considerably by incorporating the content released from the germinal vesicles (GVs). In parallel, the transferred nucleus genes must be reset and function similarly as the relevant genes in normal embryo reprogramming. This, among others, covers the relevant epigenetic modifications and the appropriate organization of chromatin in pseudo-pronuclei. While reprogramming in SCNT is often discussed, the remodeling of transferred nuclei is much less studied, particularly in the context of the developmental potential of SCNT embryos. It is now evident that correct reprogramming mirrors appropriate remodeling. At the same time, it is widely accepted that the process of rebuilding the nucleus following SCNT is instrumental to the overall success of this procedure. Thus, in our contribution, we will mostly focus on the remodeling of transferred nuclei. In particular, we discuss the oocyte organelles that are essential for the development of SCNT embryos.

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.

Long terminal repeats power evolution of genes and gene expression programs in mammalian oocytes and zygotes.

Retrotransposons are "copy-and-paste" insertional mutagens that substantially contribute to mammalian genome content. Retrotransposons often carry long terminal repeats (LTRs) for retrovirus-like reverse transcription and integration into the genome. We report an extraordinary impact of a group of LTRs from the mammalian endogenous retrovirus-related ERVL retrotransposon class on gene expression in the germline and beyond. In mouse, we identified more than 800 LTRs from ORR1, MT, MT2, and MLT families, which resemble mobile gene-remodeling platforms that supply promoters and first exons. The LTR-mediated gene remodeling also extends to hamster, human, and bovine oocytes. The LTRs function in a stage-specific manner during the oocyte-to-embryo transition by activating transcription, altering protein-coding sequences, producing noncoding RNAs, and even supporting evolution of new protein-coding genes. These functions result, for example, in recycling processed pseudogenes into mRNAs or lncRNAs with regulatory roles. The functional potential of the studied LTRs is even higher, because we show that dormant LTR promoter activity can rescue loss of an essential upstream promoter. We also report a novel protein-coding gene evolution-D6Ertd527e-in which an MT LTR provided a promoter and the 5' exon with a functional start codon while the bulk of the protein-coding sequence evolved through a CAG repeat expansion. Altogether, ERVL LTRs provide molecular mechanisms for stochastically scanning, rewiring, and recycling genetic information on an extraordinary scale. ERVL LTRs thus offer means for a comprehensive survey of the genome's expression potential, tightly intertwining with gene expression and evolution in the germline.

The nucleolus-like and precursor bodies of mammalian oocytes and embryos and their possible role in post-fertilization centromere remodelling.

In nearly all somatic cells, the ribosome biosynthesis is a key activity. The same is true also for mammalian oocytes and early embryos. This activity is intimately linked to the most prominent nuclear organelles - the nucleoli. Interestingly, during a short period around fertilization, the nucleoli in oocytes and embryos transform into ribosome-biosynthesis-inactive structures termed nucleolus-like or nucleolus precursor bodies (NPBs). For decades, researchers considered these structures to be passive repositories of nucleolar proteins used by the developing embryo to rebuild fully functional, ribosome-synthesis competent nucleoli when required. Recent evidence, however, indicates that while these structures are unquestionably essential for development, the material is largely dispensable for the formation of active embryonic nucleoli. In this mini-review, we will describe some unique features of oocytes and embryos with respect to ribosome biogenesis and the changes in the structure of oocyte and embryonic nucleoli that reflect this. We will also describe some of the different approaches that can be used to study nucleoli and NPBs in embryos and discuss the different results that might be expected. Finally, we ask whether the main function of nucleolar precursor bodies might lie in the genome organization and remodelling and what the involved components might be.

Nucleoli in embryos: a central structural platform for embryonic chromatin remodeling?

Nucleoli are the site of ribosomal RNA production and subunit assembly. In contrast to active nucleoli in somatic cells, where three basic sub-compartments can be observed, mammalian oocytes and early embryos contain atypical nucleoli termed "nucleolus-like bodies" or "nucleolus precursor bodies", respectively. Unlike their somatic counterparts, these structures are composed of dense homogenous fibrillar material and exhibit no polymerase activity. Irrespective of these unusual properties, they have been shown to be absolutely essential for embryonic development, as their microsurgical removal results in developmental arrest. Historically, nucleolus-like and nucleolus precursor bodies have been perceived as passive storage sites of nucleolar material, which is gradually utilized by embryos to construct fully functional nucleoli once they have activated their genome and have started to produce ribosomes. For decades, researchers have been trying to elucidate the composition of these organelles and provide the evidence for their repository role. However, only recently has it become clear that the function of these atypical nucleoli is altogether different, and rather than being involved in ribosome biogenesis, they participate in parental chromatin remodeling, and strikingly, the artificial introduction of a single NPB component is sufficient to rescue the developmental arrest elicited by the NPB removal. In this review, we will describe and summarize the experiments that led to the change in our understanding of these unique structures.

Dissecting the role of the germinal vesicle nuclear envelope and soluble content in the process of somatic cell remodelling and reprogramming.

Differentiated nuclei can be reprogrammed/remodelled to totipotency after their transfer to enucleated metaphase II (MII) oocytes. The process of reprogramming/remodelling is, however, only partially characterized. It has been shown that the oocyte nucleus (germinal vesicle - GV) components are essential for a successful remodelling of the transferred nucleus by providing the materials for pseudo-nucleus formation. However, the nucleus is a complex structure and exactly what nuclear components are required for a successful nucleus remodelling and reprogramming is unknown. Till date, the only nuclear sub-structure experimentally demonstrated to be essential is the oocyte nucleolus (nucleolus-like body, NLB). In this study, we investigated what other GV components might be necessary for the formation of normal-sized pseudo-pronuclei (PNs). Our results showed that the removal of the GV nuclear envelope with attached chromatin and chromatin-bound factors does not substantially influence the size of the remodelled nuclei in reconstructed cells and that their nuclear envelopes seem to have normal parameters. Rather than the insoluble nuclear lamina, the GV content, which is dissolved in the cytoplasm with the onset of oocyte maturation, influences the characteristics and size of transferred nuclei.

The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition.

The oocyte (maternal) nucleolus is essential for early embryonic development and embryos originating from enucleolated oocytes arrest at the 2-cell stage. The reason for this is unclear. Surprisingly, RNA polymerase I activity in nucleolus-less mouse embryos, as manifested by pre-rRNA synthesis, and pre-rRNA processing are not affected, indicating an unusual role of the nucleolus. We report here that the maternal nucleolus is indispensable for the regulation of major and minor satellite repeats soon after fertilisation. During the first embryonic cell cycle, absence of the nucleolus causes a significant reduction in major and minor satellite DNA by 12% and 18%, respectively. The expression of satellite transcripts is also affected, being reduced by more than half. Moreover, extensive chromosome bridging of the major and minor satellite sequences was observed during the first mitosis. Finally, we show that the absence of the maternal nucleolus alters S-phase dynamics and causes abnormal deposition of the H3.3 histone chaperone DAXX in pronuclei of nucleolus-less zygotes.

Clearance of Parental Products.

The beginning of development is controlled parentally. For example, early zygotic proteosynthesis produces proteins encoded by the maternal transcriptome. As parental factors become replaced by factors synthesized in the embryo, parental developmental control is gradually passed to the embryo. This chapter focuses on the clearance of parental factors during oocyte-to-embryo transition in vertebrates. Coordinated removal of parental factors erases ancestral oocyte identity of the zygote and facilitates reprogramming of gene expression into a state that will support development of a new organism. Here, we will review functional and mechanistic aspects of clearance of selected parental factors from early embryos, including different types of maternal RNAs, proteins, erasure of chromatin features of maternal and paternal genomes, as well as consumption of yolk and elimination of paternal mitochondria.

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.

Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries.

In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin. If they are removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long-standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article, we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment.

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.

Production of mouse embryonic stem cell lines from maturing oocytes by direct conversion of meiosis into mitosis.

ESCs are most commonly derived from embryos originating from oocytes that reached metaphase II. We describe here a novel approach where ESCs with all pluripotency parameters were established from oocytes in which metaphase I was converted, from the cell cycle perspective, directly into metaphase II-like stage without the intervening anaphase to telophase I transition. The resulting embryos initiate development and reach the blastocyst stage from which the ESC lines are then established. Thus, our approach could represent an ethically acceptable method that can exploit oocytes that are typically discarded in in vitro fertilization clinics. Moreover, our results also indicate that the meiotic cell cycle can be converted into mitosis by modulating chromosomal contacts that are typical for meiosis with subsequent licensing of chromatin for DNA replication.

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.

Nuclear Lamins: Key Proteins for Embryonic Development.

Lamins are essential components of the nuclear envelope and have been studied for decades due to their involvement in several devastating human diseases, the laminopathies. Despite intensive research, the molecular basis behind the disease state remains mostly unclear with a number of conflicting results regarding the different cellular functions of nuclear lamins being published. The field of developmental biology is no exception. Across model organisms, the types of lamins present in early mammalian development have been contradictory over the years. Due to the long half-life of the lamin proteins, which is a maternal factor that gets carried over to the zygote after fertilization, investigators are posed with challenges to dive into the functional aspects and significance of lamins in development. Due to these technical limitations, the role of lamins in early mammalian embryos is virtually unexplored. This review aims in converging results that were obtained so far in addition to the complex functions that ceases if lamins are mutated.