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.

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.

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.

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.

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.

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.

Chromatin acetylation in human oocytes.

OBJECTIVES: The frequency of aneuploidies in human oocytes is extremely high. It is hypothesized that the cause may be due to abnormal chromatin (histone) acetylation/deacetylation. The aim of our study was to analyzed the acetylation/deacetylation pattern in spare human oocytes. MATERIALS AND METHODS: Human spare oocytes (311), in other words oocytes that were not mature when collected from follicles or control oocytes (bovine, mouse), were fixed with paraformaldehyde and then labeled with antibodies against acetylated histones. RESULTS: Labeling against AcH4/K12 or hyperacetylated H4 showed high intensity of the fluorescence signal in all immature (germinal vesicle staged) and approximately 50% of the maturing (mature) oocytes. CONCLUSION: In conclusion, the labeling of human oocytes (chromosomes) showed very inconsistent patterns of acetylation/deacetylation, what may suggest they did not reach the metaphase II stage at the time of follicle aspiration, and were epigenetically abnormal. It may also explain the high frequency of chromosomal abnormalities in human oocytes.

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.

An observational study of assisted reproductive technology outcomes in new European Union member states: an overview of protocols used for ovarian stimulation.

BACKGROUND: The development of new fertility treatment options has facilitated individualized assisted reproductive technology (ART) protocols to improve outcomes. Manufacturing improvements to recombinant human follitropin alfa have allowed precise dosing based on mass (filled-by-mass; FbM) rather than bioactivity (filled-by-bioassay; FbIU). Continued monitoring and reporting of follitropin alfa treatment outcomes in routine clinical practice is essential. OBJECTIVE: To provide an overview of the frequency of different controlled ovarian-stimulation protocols used in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles in new European Union member states, and to provide post-registration efficacy and safety data on follitropin alfa. RESEARCH DESIGN AND METHODS: A 2-year, prospective, observational, multicentre, Phase IV study conducted at ART clinics in the Czech Republic, Estonia, Latvia, Lithuania, Poland, Slovakia and Slovenia. Women aged 18-47 years undergoing ovarian stimulation with follitropin alfa for conventional IVF or ICSI were eligible for inclusion. The main treatment outcome was cumulative clinical pregnancy rate. Data were analysed descriptively. RESULTS: Clinical pregnancy outcomes were available for 4055 of 4085 (99.3%) patients. In total, 1897 (46.8%) patients used follitropin alfa FbIU; 2133 (52.6%) used follitropin alfa FbM. Clinical pregnancy was achieved by 39.5% (1603/4055) of patients. A greater proportion of patients with polycystic ovary syndrome achieved a clinical pregnancy than those with endometriosis (41.8% vs 37.8%, respectively). A higher cumulative pregnancy rate was observed with the use of follitropin alfa FbM than follitropin alfa FbIU (41.3% vs 37.8%, respectively; p = 0.02). CONCLUSIONS: This study represents the most comprehensive audit of individualized ART in clinical practice in Central and Eastern Europe. Overall, clinical pregnancy was achieved by 39.5% of patients after stimulation with follitropin alfa. The use of follitropin alfa FbM resulted in a higher cumulative pregnancy rate than did the FbIU formulation. However, limitations of the study include the observational and non-comparative study design, and descriptive nature of statistical analyses; furthermore, the study was not designed to make direct comparisons between the success rates of different ovarian-stimulation protocols.

How to repair the oocyte and zygote?

The supply of human oocytes is very limited. This restricts not only certain assisted reproduction procedures in IVF clinics where recipients wait for oocytes from donors, but also development of some promising approaches, like therapeutic nuclear transfer with subsequent derivation of patient compatible embryonic stem cells. Moreover, in some patients, collected oocytes exhibit certain specific defects, and logically, we can expect that after fertilization, the embryos arising from these defective oocytes may not develop or that their development might eventually be compromised. For this reason, an increased effort to determine how to repair oocytes is evident in the literature. In general, abnormalities (defects) can be detected in different oocyte components, the zona pellucida, cytoplasm, nucleus (chromosomes) and nucleolus. Whereas defects of a nuclear component are impossible (nuclear DNA) or very hard to repair (nucleolus), zona pellucida abnormalities and cytoplasm defects (for example, if containing mutated mitochondrial DNA, mtDNA) can be repaired in some cases with the help of micromanipulation schemes. In the present article, we will briefly outline the current methodological approaches that can be used to repair the oocyte or one-cell stage embryo.