Marcella Marino

Chaotic mosaicism in human preimplantation embryos is correlated with a low mitochondrial membrane potential.

OBJECTIVE To determine the relationship between the intrinsic mitochondrial deltapsi of human embryos and the embryo karyotype. DESIGN Analysis of mitochondrial deltapsi of living embryos followed by chromosomal enumeration with fluorescence in situ hybridization. A tertiary center for assisted reproduction technology. PATIENT(S) Fifty-two patients attending the fertility center for assisted reproduction. INTERVENTION(S) Donated embryos were loaded with a mitochondrial deltapsi-sensitive fluorescence dye. MAIN OUTCOME MEASURE(S) Mitochondrial deltapsi was measured by confocal microscopy. Subsequently, embryos were fixed and fluorescence in situ hybridization analysis was used to denote embryo karyotype. MAIN OUTCOME MEASURE(S) Mitochondrial deltapsi and embryo karyotype. RESULT(S) An association was observed between low mitochondrial deltapsi and the detection of chaotic mosaicism. Analysis of oocytes suggested that this was due to the effect of low mitochondrial deltapsi on the morphology of the meiotic apparatus. CONCLUSION(S) The data suggest that the intrinsic mitochondrial deltapsi of human oocytes programs the developmental fate of embryos through an effect on the ability of oocytes to form a normal meiotic apparatus and not through nondisjunction.

Sex determination of buffalo embryos ( Bubalus bubalis ) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).

Interaction of cell cycle kinases, microtubules, and chromatin in ascidian oocytes during meiosis.

We used kinase assays and confocal microscopy to study the interaction of cell cycle proteins with microtubule organising centres (MTOC) and chromatin in ascidian oocytes during meiosis. The activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) appear not to be correlated in control oocytes. MPF activity peaks during metaphase I and II of the meiotic cell cycle whereas the activity of MAPK peaks at telophase I and is subsequently degraded to remain at low levels for the remainder of meiosis. The protein synthesis inhibitor emetine induces the degradation in MPF activity in unfertilized metaphase‐I (M‐I) oocytes, while MAPK is unaffected. Emetine does not alter the activities of these cell cycle kinases in fertilized oocytes during meiosis I but MPF activity remains low while MAPK activity is high for an elongated time period and oocytes do not complete meiosis I. Emetine induces maternal MTOC duplication in unfertilized M‐I oocytes and prevents sperm aster growth in fertilized oocytes, but it does not alter the M‐I meiotic apparatus in unfertilized oocytes. These experiments suggest that neither MPF alone nor emetine‐sensitive proteins are responsible for M‐I arrest in ascidian oocytes, MAPK may ensure this stability. In addition, we showed that the maternal MTOC is present at M‐I but suppressed from duplicating in an emetine‐sensitive manner. Mol. Reprod. Dev. 56:155–162, 2000.

Meiosis-associated calcium waves in ascidian oocytes are correlated with the position of the male centrosome.

We have used confocal microscopy to measure calcium waves and examine the distribution of tubulin in oocytes of the ascidian Ciona intestinalis during meiosis. We show that the fertilisation calcium wave in these oocytes originates in the vegetal pole. The sperm penetration site and female meiotic apparatus are found at opposite poles of the oocyte at fertilisation, confirming that C. intestinalis sperm enter in the vegetal pole of the oocyte. Following fertilisation, ascidian oocytes are characterised by repetitive calcium waves. Meiosis I-associated waves originate at the vegetal pole of the oocyte, and travel towards the animal pole. In contrast, the calcium waves during meiosis II initiate at the oocyte equator, and cross the oocyte cytoplasm perpendicular to the point of emission of the polar body. Immunolocalisation of tubulin during meiosis II reveals that the male centrosome is also located between animal and vegetal poles prior to initiation of the meiosis II-associated calcium waves, suggesting that the male centrosome influences the origin of these calcium transients. Ascidians are also characterised by an increase in sensitivity to intracellular calcium release after fertilisation. We show that this is not simply an effect of oocyte activation. The data strongly suggest a role for the male centrosome in controlling the mechanism and localisation of post-fertilisation intracellular calcium waves.

Nicotinamide alters the calcium release pattern and the degradation of MPF activity after fertilisation in ascidian oocytes.

Fertilisation in ascidian oocytes triggers a plasma membrane current, the release of intracellular calcium and the degradation of Maturation Promoting Factor (MPF) activity leading to the completion of meiosis and the initiation of embryo development. We have previously shown that the fertilisation current in ascidians is produced through the metabolism of nicotinamide nucleotide (NN) metabolites to ADP ribose. In this study we have used nicotinamide to test whether NN metabolism plays additional roles in fertilisation in ascidians. Nicotinamide treatment blocked calcium-induced calcium release (CICR) and arrested the cell cycle prior to the completion of meiosis I. Nicotinamide further prevented the abolition of MPF activity after fertilisation. Interestingly, nicotinamide treatment caused ascidian oocytes to form interphase-like pronuclei after fertilisation, despite the high MPF activity. The data demonstrate that NN metabolism is involved in calcium signalling through CICR and further suggest that a NN metabolite acts as a messenger connecting MPF activity to the formation of the meiotic apparatus.

Meiotic Cell Cycle Control by Mos in Ascidian Oocytes

Mitotic cell division cycle is regulated by several control mechanisms generally known as checkpoints, whose main function is to ensure that critical events in the cell division such as DNA replication and chromosome segregation occur with high fidelity and in the correct order and time. A recent view of cell cycle regulation, indicate that checkpoints work as signal transduction pathways with their initiating signals, sensors, transducers, and effectors (Elledge, 1996). Similarly, meiotic cell cycle regulation have specific checkpoints and two of them have been recently characterized at molecular level. The first ensures the completion of recombination before the formation of meiosis I spindle; the second blocks anaphase I until all the paired chromosomes are correctly attached to the spindle (Page and Orr-Weaver, 1997). However, the most intriguing difference between mitosis and meiosis respect to cell cycle regulation, is the ability of animal oocytes to arrest at specific stage during maturation, and maintain this block for extremely long time: from years (frog) to decades (human) (Sagata, 1996a,b). It is universally accepted that the meiotic block is due to the activity of a cytostatic factor (CSF) primarily identified by Masui (Masui and Markert, 1971).

Activation of Ciona Intestinalis at Fertilisation is Controlled by Nicotinamide Nucleotide Metabolism

Fertilisation is characterised by an ion current across the oocyte plasma membrane (DeFelice et al., 1986), a transient increase in intracellular calcium (see Jaffe, 1985), and the release of the cell cycle block controlled at the level of MPF (Newport and Kirschner, 1984). The fertilisation calcium signal appears to trigger the resumption of the cell cycle at fertilisation in oocytes arrested at meiosis II or after the completion of meiosis (reviewed in Whitaker and Patel, 1990). However, the role of intracellular second messengers, including calcium, in the control of progression through the early meiotic cell cycle, is less well defined (see Whitaker and Patel, 1990; Whitaker, 1996).