Embryos undergo chaotic division and decrease in quality on day 3 with a reduction in the rates of subsequent blastocyst formation. Disordered cleavage causes a deterioration in embryonic quality, here we assessed the relationship between an cleavage model in first mitosis and the chromosomal status of human embryos, and discuss the potential biological and clinical implications for the cleavage model as a single parameter that can be used to assess embryonic quality. Thirty-two infertile couples, with normal karyotypes and who underwent their first IVF cycle were recruited to donate one normal two-cell-stage embryo each for this study between 2019 and 2020. Twenty-eight two-cell embryos underwent preimplantation genetic testing of each blastomere, and four chaotic-division embryos were stained with Hoechst and cultured in a confocal laser-scanning microscopy incubator system. This system showed high specificity and PPV but low sensitivity and NPV using the CM in the prediction of euploidy, indicating that CM could be considered a screening method for embryo selection; additional observational studies using the CM to select transferable embryos are needed before it can be used in clinical practice.

Changes in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated egg cells and fusion products of pairs of one egg and one sperm cell of Zea mays L. have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'dihexyloxa-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjuction with computer-controlled video image analysis. In addition, confocal laser scannig microscopy has been used in conjuction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetra cycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+ in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+ in cytoplasm is also discrnible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of Cam in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Following in vitro fertilisation of single isolated eggs substantial changes in the Ca2+ levels occur which include an increase in the membrane Ca2+ of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products. Compared with isolated egg cell protolasts an increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the fluorescene around the fusion product is visible after 16 h in in culture. The sperm cell chromatin in the fusion product is highly condensed, unlike that of the egg cell, and confocally imaged serial optical sections of the in vitro fusion product reveal the occurrence of karyogamy 35 min following gamete fusion. First visual evidence for intermingling of sperm nuclear chromatin in the zygotic nuclei is also provided.

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

Alterations in the rate of oocyte meiotic maturation (OM) and the timing of the metaphase-anaphase transition may predispose oocytes to premature centromere separation (PCS) and aneuploidy. Tamoxifen has the potential for perturbing the rate of OM since it can function as a calcium antagonist by binding to calmodulin and inhibiting the formation of a calcium-calmodulin complex which is needed for activating calmodulin-dependent cAMP phosphodiesterase and initiating OM. The objective of this study was to test the hypothesis that tamoxifen alters the rate of OM and predisposes oocytes to PCS and aneuploidy. Different does of tamoxifen were administered by oral gavage to female mice preovulation. Metaphase II oocyte and 1-cell zygote chromosomes were C-banded and cytogenetically analysed. Tamoxifen treatment resulted in a modest, but significant (p < 0.05), increase in oocytes with PCS. Similar frequencies of hyperploidy and oocytes with unpaired, single chromatids (SC) were found. Metaphase I, diploid and premature anaphase (PA) oocytes were not detected. Hyperploidy, polyploidy, PCS, PA and SC were not detected in zygotes. These data indicate that the levels of tamoxifen-induced PCS found in mouse oocytes did not predispose zygotes to aneuploidy. Tamoxifen did, however, reduce the proportion of females exhibiting oestrus.

Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading — which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM) — and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl−). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.