Fertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes – most probably a product of controlled ovarian stimulation – are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development.

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

The outer surface of hamster zona pellucida has a strong ability to induce the sperm acrosome reaction, whereas neither the inner surface of the zona nor the oolemma has this ability. Therefore, the spermatozoa that fertilise oocytes after subzonal insemination of spermatozoa (SUZI) must have been either acrosome-reacted prior to injection or acrosome-reacted spontaneously while swimming within the perivitelline space. The outer surface of fresh mouse zona is not capable of inducing the acrosome reaction of hamster spermatozoa. When fixed with glutaraldehyde, however, it became capable of inducing the hamster sperm acrosome reaction rather efficiently. The reason for this is not clear.

The intracellular pH of the mouse egg was measured during fertilisation to determine whether an increase in pH accompanies activation of this mammalian egg. The pH-sensitive dye BCECF [2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein] was introduced into the mouse egg by incubation in BCECF-AM or by microinjection of dextran-conjugated BCECF. The cells were also loaded with the DNA-specific fluorochrome Hoechst 33342 to confirm fertilisation by observation of Hoechst-stained, decondensing sperm heads in the cytoplasm. The ratio of emission intensities for the dye (494/440 nm excitation wavelengths) was monitored continuously with a photon-counting photomultiplier tube. There was no change in pH during or after fertilisation. Control eggs displayed the expected increase in pH when exposed to NH4C1. In other experiments, intracellular pH and intracellular Ca2+ were monitored simultaneously during fertilisation. The eggs were injected with BCECF dextran and Fura dextran. Fluorescence emission was recorded at excitation wavelengths of 495 nm (BCECF, pH-sensitive wavelength) and 385 nm (Fura, Ca2+-sensitive wavelength). A decrease in emission intensity at 385 nm excitation clearly marked the repetitive Ca2+ transients at egg activation. There was no change in the fluorescence emitted at 495 nm excitation, indicating an absence of any change in intracellular pH. These results indicate that intracellular alkalinisation of the cytoplasm does not accompany activation of this vertebrate egg.

Under our culture conditions, mouse embryos from the BALB/c inbred mouse strain develop successfully in culture only from the late 2-cell stage onwards (so-called 2-cell block), whether or not EDTA is added to the culture medium. (CBA × C57BL) F2 embryos do not exhibit a 2-cell block. Medium conditioned by culture of non-blocking embryos from the 2-cell to the 8-cell stage did not improve the development of blocking embryos, nor did co-culture of blocking and non-blocking embryos, with or without conditioned medium. On the other hand phytohaemagglutinin (PHA)-assisted aggregation of an early 2-cell BALB/c embryo with five surrounding non-blocking F2 embryos (2-cell or 8-cell) or five BALB/c 8-cell embryos allowed the early 2-cell BALB/c embryos to develop into blastocysts within 72 h. Aggregation of blocking BALB/c 2-cell embryos with each other had no ‘rescue’ effect. When blocking and non-blocking 2-cell embryos were aggregated together, an integrated blastocyst was formed; but when the early 2-cell BALB/c embryos were aggregated with non-blocking 8-cell embryos, the blocking embryos formed a separate small blastocyst, which nonetheless retained adherent contact with the non-blocking embryos throughout the culture period. Ultrastructural analysis showed that 2-cell embryos aggregated with the aid of PHA form close adherent cell contacts up to several micrometres in length.

The molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 μm, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 μm, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3–4 mm) and large antral follicles (5–6 mm) had mean oocyte diameters of 115 and 119 μm respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 μm) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 μm) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.

Taurine and hypotaurine seem to be important compounds for sperm survival and capacitation, the fertilisation process and embryo development, and are present in both sperm and genital secretions. Hypotaurine has protective effects against peroxidative damage. We have established a simple method for hypotaurine quantification in sperm and genital secretions. The mean concentration of hypotaurine is significantly higher in bovine than in human spermatozoa and in seminal plasma. We observed that both molecules are secreted by cow, sow, goat and rabbit oviduct epithelial cell monolayers. In rabbit the release is ascorbic acid dependent. Goat oviduct epithelial cells are able to use the transsulfuration pathway to form hypotaurine and taurine from methionine. We were able to identify cysteine sulfinate decarboxylase (EC 4.1.1.29) activity in cow and goat tubal monolayers. Our results demonstrate that hypotaurine and taurine are secreted by oviduct epithelium, and synthesised by tubal cells via the cysteine sulfinic acid pathway. The data obtained emphasise the importance of hypotaurine and taurine for gamete maturation, fertilisation and early embryonic development.

Cumulus-enclosed or cumulus-free pig oocytes were cultured for 48 h in a serum-free maturation medium, TCM-199B containing 1 mg/ml polyvinylalcohol, supplemented with gonadotropins (PMSG + hCG) and/or epidermal growth factor (EGF), and inseminated with frozen-thawed ejaculated spermatozoa. Fourteen hours after insemination, the ability of oocytes to form male pronucleus (MPN) was examined. EGF significantly increased the ability of cumulus-enclosed oocytes to form MPN irrespective of the concentrations (1–100 ng/ml) tested. Gonadotropins had a greater effect than EGF on MPN formation in cumulus-enclosed oocytes, and a synergetic effect was observed when EGF and gonadotropins were added together. However, neither EGF nor gonadotropins exhibited a stimulatory effect on MPN formation in cumulus-free oocytes. These results indicate that the role(s) of both EGF and gonadotropins in stimulating cytoplasmic maturation of pig oocytes is mediated by cumulus.

In the starfish Asterias amurensis, the jelly coat of the eggs contains a glycoprotein essential for the induction of the acrosome reaction in homologous spermatozoa that is termed the acrosome-reaction-inducing substance (ARIS).ARIS is a highly sulphated and fucose-rich glycoprotein of extremely high molecular mass(>104 kDa). ARIS was irradiated with high-energy eletrons in order to estimate the minimum size required for its biological activity. The minimum functional unit or target size of ARIS was estimated to be c. 14 kDa by target size analysis. ARIS was significantly disintegrated by the irradiation, yet the total sugar content was not apparently reduced. The binding of 125I-labelled ARIS to spermatozoa competed with that of irradiated ARIS, although the affinity of ARIS was much reduced after irradiation.

The effect of temperature fluctuation on spindle integrity and chromosomal organisation in the human oocyte, and the consequences of such effects on the chromosomal constitution of resulting parthenotes, were investigated. A total of 340 oocytes were stained immunocytochemically with an antibody to α-tubulin, and 502 were activated parthenogenetically. Exposure of oocytes to room temperature for 2, 10 or 30 min caused disruption of the spindle in 77%(n = 26),72%(n = 18) and 89% (n = 19) of cases respectively, with evidence of chromosomal dispersal in 50%,56% and 52.6% respectively. These effects were reversed when oocytes were returned to 37°C after exposure to room temperature for 2 min, but not after 10 min or 30 min: Temperature reduction affected rates of parthenogenetic activation of oocytes (2 min: 67%, n = 27; 10 min: 68%, n = 28; 30 min; 54%, n = 35) and cleavege of resulting parthenotes, but only if oocytes were exposed to room temperature for 30 min (30 min: 53%, n = 19).There is a direct association between temperature-induced spindle damage in the oocyte(70%, 50 of 63) and chromosomal abnormalities in parthenotes developed from oocytes exposed to room temperature (56%,23 of 41; P < 0.01).