Elisabetta Tosti

Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays

PurposeTo determine the level of expression for mRNAs that regulate DNA repair activity in oocytes at the germinal vesicle (GV) stage. Reactive oxygen species (ROS) have been shown to play a major role in the appearance of deleterious DNA decays, and this study focuses on the repair of damage linked to decay caused by the action of ROS. The oocyte needs a mechanism for repairing DNA decays in the early preimplantation embryo before the onset of genomic activation, since in the absence of repair, residual DNA damage would lead to either apoptosis or tolerance. Tolerance of DNA damage is a source of potential mutations.MethodGV oocytes were selected for this study, both for the ethical reason that they are unsuitable for patient treatment, and because no transcription takes place during the period from GV to MII and then prior to genomic activation. The GV oocyte is therefore a good model for looking at DNA during the first cleavages of early preimplantation development. Six cohorts of GV oocytes were pooled for extraction of mRNA; the DNA was analysed using Affimetrix HG-UG133 Plus 2, containing 54,675 probe sets; spike and housekeeping genes were also added as internal controls.ResultsIn GV oocytes, DNA repair pathways for oxidized bases are redundant. One step repair procedure (OSR), BER (base excision repair), MMR (mismatch repair) and NER (Nucleotide excision repair) are present. All the recognition proteins are also present. The chromatin assembly factors necessary for the maintenance of genomic stability are highly expressed.ConclusionGene expression analysis shows that the oocyte does not allow a high level of tolerance for DNA decays. This regulatory mechanism should avoid transmitting mutations into the next generation.

Developmental Potential in Bovine Oocytes Is Related to Cumulus-Oocyte Complex Grade, Calcium Current Activity, and Calcium Stores

Abstract A morphological classification of the immature cumulus-oocyte complex (COC), which grossly resembled the atresia grade of its follicle source, was used in bovine oocytes to determine 1) the developmental potential by either in vitro fertilization or parthenogenetic activation, 2) the calcium current activity by whole-cell voltage clamp technique, and 3) the intracytoplasmic calcium stores by microfluorimetric evaluation. The COC classification took into account some cumulus and ooplasm features, designated as follows: A) presence of a clear and compact cumulus and translucent ooplasm, B) dark and compact cumulus and dark ooplasm, and C) dark and expanded cumulus and dark ooplasm. We found no difference between in vitro fertilization and parthenogenetically activated oocytes in terms of cleavage rate and blastocyst production. Both protocols indicated a significant variability between the three compared COC categories. The B-COCs showed the highest embryo production efficiency as well as the greatest Ca2+ current activity, whereas A-COCs showed an opposite pattern. The C-COCs, mostly attributed to atretic and heavily atretic follicles, showed morphological characteristics between those of A- and B-COCs. Stores of Ca2+ were significantly greater in A-COCs than in B- and C-COCs in the case of immature oocytes, and greater in B-COCs than in C-and A-COCs in the case of in vitro-matured oocytes. These results demonstrate that in the bovine 1) the considered morphological criteria for oocyte classification are related to developmental competence, 2) plasma membrane Ca2+ current in the immature oocyte is related to developmental potential, and 3) calcium stores are related to morphological quality in immature oocytes and to developmental competence in mature oocytes.

A morphological and functional study of fusibility in round-headed spermatozoa in the human

OBJECTIVE To study the molecular origin and functionality of the plasma membrane of round-head spermatozoa in the human. DESIGN Clinical and laboratory study. SETTING Patients in a clinical and academic environment. PATIENTS Men with round-head spermatozoa. RESULTS Pisum sativum lectin homogeneously stains the surface of round sperm; however, the staining pattern and transmission electron microscopy show that the plasma membrane does not alter after exposure to the calcium ionophore A23187. In a clinical program, round-head spermatozoa injected subzonally into metaphase II oocytes with or without pretreatment with the fusogen polyethylene glycol did not bind or fuse to the oocyte surface. CONCLUSION The data suggests that plasma membrane fusion in human gametes is regulated by specific surface molecules and that exposure of these molecules on the sperm surface cannot be triggered by elevating intracellular calcium alone.

The impact of in vitro fertilization on health of the children: an update

Infertile couples make up approximately 10% of the worldwide population, and around 1% of current live births are a result of assisted reproductive technology (ART). Since the time that this technology was first applied, many studies have been performed in order to determine the risk associated with infertility treatments. Short- and long-term risks have been identified, which confirm that the major complications are due to multiple pregnancies. In a previous study we described in detail the main reproductive processes, the techniques for in vitro fertilization (IVF) and the risks associated with each of them, with a focus on intracytoplasmic sperm injection (ICSI). In this review we provide an update from 2007 to the present. In particular, in addition to new information on post-pregnancy complications and infant morbidity and malformations, we report data on rare syndromes, including recent case reports. Although data are controversial, an association between IVF and a minor increase in the incidence of birth defects has been confirmed. Several lines of evidence also suggest that there may be a link between ART and psychological disorders in the parents and the child. Finally, recent findings draw attention to the need for accurate clinical and psychological counselling of couples before any treatment decisions are made.

A soluble sperm factor gates Ca2+-activated K+ channels in human oocytes

AbstractPurpose: Our goal was to study the activation current in physiologically competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa or aged in vitro, and, in particular, to determine whether a soluble sperm factor triggers a fertilization current comparable to that observed with intact spermatozoa and to characterize the current involved. Methods: The whole-cell voltage-clamp technique was used on spare metaphase II oocytes, obtained with patient consent from IVF programs. In this configuration a soluble fraction from human spermatozoa was microinjected, and the current recorded. Results: Metaphase II human oocytes generate bell-shaped outward currents of 400–1000 pA (X=706±322;n=10), following injection of a cytosolic extract from human spermatozoa. The amount of sperm extract injected was less than 10% of the total oocyte volume and was equivalent to 1–10 spermatozoa. A similar current was generated following exposure to 20 µM of the calcium ionophore A23187 (n=10). The steady-state conductance of the oocyte increased from 10 to 19.8 nS (n=10) following injection of the sperm factor and from 5.3 to 27.7 nS following ionophore exposure. Both sperm factor- and ionophore-induced currents were reduced in amplitude when the unfertilized oocyte was preexposed to 25–75 µM iberiotoxin (n=8) and eliminated at a concentration of 100 µM iberiotoxin. Conclusions: The data support the hypothesis of a soluble sperm factor involved in the activation of human oocytes and shows that the initial activation response in the human oocyte is the gating of Ca2+-activated K+ channels.

Voltage Clamp of the Nuclear Envelope

By using conventional intracellular microelectrodes, we show that the starfish germinal vesicle in situ has a resting potential of +4 mV to +30 mV with respect to the cytoplasm. Patch-clamp electrodes reveal that the surface of the isolated nucleus contains ion channels of 120 pS single-channel conductance. Under whole-cell voltage-clamp conditions, the starfish germinal vesicle generates asymmetric time-variant currents that depend on the ionic environment. Fluorescent dyes in the whole-cell clamp electrode fill the nucleus, showing that the time-variant and the steady-state conductances are properties of the outer and inner nuclear membranes or something that spans them. Because scanning electron microscopy shows a density of nuclear pores in the range 60—110 per square micrometre, and the steady-state resistance in the range is 30—100 MΩ, it seems probable that the pores are not freely permeable to ions. Furthermore, fluorescent dyes of molecular weight up to 500 Da micro-injected either into the cytoplasm or the nucleus of intact oocytes accumulate in the nucleus. Our results show that the nucleus acts as an electrically isolated compartment that separates charged low molecular mass molecules including inorganic ions.

Gamete activation: basic knowledge and clinical applications

Background The first clues to the process of gamete activation date back to nearly 60 years ago. The mutual activation of gametes is a crucial event during fertilization. In the testis and ovaries, spermatozoa and oocytes are in a state of meiotic and metabolic quiescence and require reciprocal signals in order to undergo functional changes that lead to competence for fertilization. First, the oocyte activates sperm by triggering motility, chemoattraction, binding and the acrosome reaction, culminating with the fusion of the two plasma membranes. At the end of this cascade of events, collectively known as sperm capacitation, sperm-induced oocyte activation occurs, generating electrical, morphological and metabolic modifications in the oocyte. Objective and rationale The aim of this review is to provide the current state of knowledge regarding the entire process of gamete activation in selected specific animal models that have contributed to our understanding of fertilization in mammals, including humans. Here we describe in detail the reciprocal induction of the two activation processes, the molecules involved and the mechanisms of cell interaction and signal transduction that ultimately result in successful embryo development and creation of a new individual. Search methods We carried out a literature survey with no restrictions on publication date (from the early 1950s to March 2016) using PubMed/Medline, Google Scholar and Web of Knowledge by utilizing common keywords applied in the field of fertilization and embryo development. We also screened the complete list of references published in the most recent research articles and relevant reviews published in English (both animal and human studies) on the topics investigated. Outcomes Literature on the principal animal models demonstrates that gamete activation is a pre-requisite for successful fertilization, and is a process common to all species studied to date. We provide a detailed description of the dramatic changes in gamete morphology and behavior, the regulatory molecules triggering gamete activation and the intracellular ions and second messengers involved in active metabolic pathways in different species. Recent scientific advances suggest that artificial gamete activation may represent a novel technique to improve human IVF outcomes, but this approach requires caution. Wider implications Although controversial, manipulation of gamete activation represents a promising tool for ameliorating the fertilization rate in assisted reproductive technologies. A better knowledge of mechanisms that transform the quiescent oocyte into a pluripotent cell may also provide new insights for the clinical use of stem cells.

Ca2+ signaling during maturation of cumulus-oocyte complex in mammals.

Under the influence of gonadotropins or growth factors, a close cooperation develops between cumulus cells and the oocyte that is implicated in transmitting signals involved in maintaining or releasing the meiotic arrest in the oocyte. While cyclic adenosine 5′‐monophosphate (cAMP) is a key molecule in maintaining the meiotic arrest, calcium (Ca2+) may play a role in controlling either spontaneous or gonadotropin‐induced oocyte maturation, possibly by modulating intracytoplasmic cAMP concentrations via Ca2+‐sensitive adenylate cyclases. This review focuses on the mechanisms related to the origin of the Ca2+ wave that travels from the cumulus cells to the oocyte, and discusses the source of variations affecting the dynamics of this wave. Mol. Reprod. Dev. 78:744–756, 2011.

Is the plasma membrane of the human oocyte reorganised following fertilisation and early cleavage

The purpose of the present study was to determine whether the plasma membrane of the human oocyte is reorganised following fertilisation and during early cleavage. In order to characterise and localise the major sugar moieties on surface glycoproteins, oocytes and embryos were labelled with a range of fluorescent lectins. Regional organisation of plasma membrane microvilli in oocytes and embryos was also studied using scanning electron microscopy (SEM). The plasma membrane of human oocytes, zygotes and early blastomeres stained strongly and homogeneously with concanavalin A and Triticum vulgaris lectin (WGA), indicating the presence of plasma membrane glycoconjugates with alpha-D-mannosyl residues, sialic acid and beta-NAc-glucosaminyl groups. We did not observe regional domains in oocytes and zygotes, suggesting that the plasma membrane is not topographically reorganised following fertilisation. SEM shows the surface of the human zygote to be organised into short microvilli 0.2-3.0 microns in length and at a density of 5-20/microns2. In early cleavage stages the microvilli are shorter and less frequent (0.2-1.0 microns; 1-5/microns2); however, there is no evidence of polarisation at this level of organisation, at either stage of development. The surface of cell fragments, common in the human embryo in vitro, differs in having few microvilli and numerous cytoplasmic blebs. In conclusion, there are no obvious morphological signs of regionalisation in the plasma membrane of the human embryo before the 8-cell stage.

Sperm nucleus decondensation, hyaluronic acid (HA) binding and oocyte activation capacity: different markers of sperm immaturity? Case reports

During the early time of IVF, sperm competence was defined as the ability to fertilize an oocyte. However, with the advent of ICSI, despite the capacity to reach 2–4cell stage, it is impossible for numerous patients, to establish a pregnancy. Instead the consensus now is that male fertility has to be defined as the capacity to produce sperm cells able to establish a full term pregnancy [1–3] Basic parameters such as concentration, motility and morphology are of limited value in determining sperm capacity to allow full embryonic development to term. Determination of DNA changes like fragmentation and condensation are independent parameters [2] and obviously of major importance. DNA fragmentation, related or not to reactive oxygen species (ROS) insult is only one piece of the problem. Indeed, sperm chromatin tertiary structure seems to be critical for correct epigenetic regulation and maintenance, and further on for male fertility [4, 5]. During very early embryogenesis, an adequate chromatin structure is also necessary for the very first cleavages [6–9]. Methylation is one of the most important regulators of the tertiary structure and imprinting; it affects sperm DNA and histones packaging it; in this way a correct recycling of homocysteine is mandatory during spermatogenesis and embryogenesis. The sperm is not just a carrier of paternal genome: it has a relevant epigenetic contribution. A defective wrapping of DNA is often linked to immaturity. Hyaluronic acid (HA) binding ability has been proposed as a tool to select the more mature spermatozoa having reached their final nuclear and cytoplasmic maturation [10, 11], even if controversies exist [12], and it is used as well in veterinary medicine [13]. In this case report we have tested the relation between HA binding and methylation effector supplementation, by comparison to what we have observed with such a supplementation for patients with poor sperm condensation.