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.

The course of the cortical reaction in the Platynereis dumerilii egg is described from live observation and from sectioned fixed material and is found to differ in several aspects from the course of cortical reactions in better-known systems. Cortical granules are unusually numerous. They are discharged by exocytosis during a period of about 25 min following fertilisation (18°C). Most of the surplus membrane material brought to the egg surface by exocytosis is set free into the perivitelline space. Swelling of egg jelly precursor secreted by cortical granule exocytosis may be causal for the detachment of the vitelline envelope from the egg cell surface which, however, remains attached punctately to the vitelline envelope by about 30000 microvilli. Under the strain of the distending vitelline envelope, the bases of the microvilli move and line up, pulling the cell surface into a network of ridges. The grooves in between the ridges are the sites of exocytoses. Cytochalasin B, generally destabilising actin filaments, induces rupture of the microvilli and exaggerated distension of the vitelline envelope during the cortical reaction. In a final phase of the cortical reaction the vitelline envelope wrinkles and falls back onto the egg cell surface, the microvilli shorten and the egg cell transiently becomes deformed by local contractions. The cortical reaction in the nereid egg is discussed as a process of distortion and reorganisation of the egg cortex and plasmalemma. The abundance of cortical granules accommodating egg jelly precursor in the Platynereis oocyte is attributed to the mode of so-called diffuse oogenesis characteristic of nereids, i.e. of differentiation of oocytes freely suspended in the coelomic fluid. In nereids, egg jelly therefore forms after fertilisation as opposed to ovulation.

Interspersed RNA is an abundant class of cytoplasmic poly(A)+ RNA which contains repetitive elements within mostly heterogeneous single copy sequences. In spite of its quantitative importance in oocytes or eggs (two-thirds of the total poly(A)+ RNA), very little is known about its synthesis, its interaction with other molecules, and its functional significance. Here, we analysed a prevalent family of interspersed RNa (XR family) during Xenopus oogenesis. We found that XR interspersed RNA, unlike extracted interspersed RNA, did not form RNA duplexes in vivo. Im small oocytes (stage III), XR RNA interacted with proteins forming rapidly sedimenting ribonucleoprotein particles (RNPs) with a median sedimentaion constant of 80S. However, towards the end of oogenesis (stage VI), these XR RNPs changed into smaller particles with a median sedimentaion constant of 40S. By analysing the proteins associated with XR RNA sequence, we have identified a 42 kilodalton protein in small oocytes, which was replaced by a 45 kilodalton protein at stage V of oogenesis.

We describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 ×106 clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.

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.

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy-ethane-N,N,N',N' -tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probaly dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30–40s before the cortical contraction is triggered by fertilisation or ionomycin- induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium the triggered by te sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation eggs appears to involve two independent pathways involving calcium.

The ability of mouse oocytes to become activated after exposure too the calcium ionophore A23187 has been investigated at different stages of meiotic maturation. The potential to respond to ionophore has been studied in relation to the time since resumption of meiotic maturation, the chromosomal conformation of the DNA within each cell and the protein synthetic profile of the maturing oocyte. Our studies demonstrate that when maturing oocytes from an MF1 strain of mice were treated with A23187 activation occured only in oocytes which had reached second meiotic metaphase (MII). However, development of the ability to respond to ionophore was not dependent on an orderly progression through normal chromosomal rearrangements such as separation at metaphase I (MI) and subsequent polar body extrusion, since there process could be prevented and the capacity to be activated became apparent in such oocytes at a time when control cells had reached MII. These data suggest that the ability to respond to ionophore depends on the development of a cytoplasmic or complex capable of monitoring the time since initiation of germinal vesicle breakdown. Metabolic radiolabelling of oocytes which were able to respond to calcium ionophore, even though they had been prevented from undergoing normal chromosomal rearrangements, showed them to be synthesising a group of proteins known as the 35 kDa complex.

Fresh and aged unfertilised human oocytes were activated by electroporation and by exposure to isotonic solution of mannitol supplemented with low concentrations of calcium magnesium and chloride. Over 95% of the fresh oocytes were activated, all showing formation of one pronucleus and extrusion of the second polar body. Oocytes activated 1 and 2 days post-collection showed activation rates of 66.6% and 64.1%, respectively; however, the proportion of one-pronucleate oocytes in these groups was significantly lower (61.6% and 23.5%, respectively). There was no difference in the activation efficiency between the two activation modes. Twelve activated oocytes from the freshly collected group cleaved when left in culture. It is concluded that, in the human, a brief exposure to isotonic solution of mannitol with low concentrations of calcium, magnesium and chloride is a very effective activation stimulus.

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.

Mast cells of connective tissue type are scattered in the interstitial compartment of the lizard Podarcis s. sicula. Their number varies during the year, showing peaks in spring and in winter, respectively.Thermal manipulation affects mast cell number (MCN): high temperature decreased MCN in both January and May, while low temperature increased MCN only in January. Ethane dimethane sulphonate, a toxin which specifically destroys Leydig cells, induced an increase in MCN on days 3 and 7 of treatment. Oestradiol treatment provoked a strong increase in MCN that was blocked by tamoxifen. Blocking androgen receptors with cyproterone acetate resulted in an increase in MCN, while testosterone injection provoked a strong decrease. These results suggest a relationship between the presence of mature Leydig cells and mast cell proliferation and/or differentiation.

Sperm plasma membrane (PM) proteins that demonstrate affinity for egg PM preparations have the potential to be biologically important during sperm-egg binding and/or fusion. In this study four such proteins have been identified. To provide quantitative evidence for possible biological function, the large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins. An aliquot from each of 24 porcine ejaculates was evaluated by the zona-free hamster ova bioassay and the remainder processed to yield sperm PM vesicles. Aliquots of sperm PM were solubilised, separated by SDS-PAGE, western blotted and probed with partially purified, biotinylated egg PM protein. Bound egg PM proteins were visualised on western blots by an avidin/biotin/horseradish peroxidase system and analysed by scanning laser densitometry. Four sperm PM proteins (62, 39, 27 and 7 kDa estimated molecular mass) were the predominant binders of egg PM. The amount of egg PM bound to the 62 kDa protein was significantly correlated with the ability of sperm from the 24 ejaculates to penetrate zona-free hamster ova (percentage of ova penetrated, p = 0.01, R = 0.65; number of penetrated sperm per ovum, p = 0.02, R = 0.63)

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.

In the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface of Spodoptera frugiperda (Sf9) cells. Expression of CD4 on Sf9 cells infected with autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhension was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.

In Scutigera coleoptrata, double spermatogenesis (macro and micro germinal cells) was reinvesti gted by means of both image analysis and electron microscopy. Image analysis of the cell-lines showed no differences in DNA content between the two types of spermatogesis.The only difference found, with the exception of the size and the related number of organelles, was that in the macro-cell line the chromatin was well dispersed and no condensed, which is the opposite of what was observed in the macro-cell line. It is asumed that the level of activity related to this condensation process is at the origin of the two pathways of development.

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.

To investigate the involvement of mitogen-activated protein kinase(MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using basic protein(MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0–20 h of culture. An abrupt increase was observed at metaphase I(30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

Uvomorulin (E-cadherin) is the major cell adhesion molecule responsible for intercellular adhesion in early mouse embryos. In contrast to other cell adhesion molecules, it is not detectable on the cell surface until around 6 h after fertilisation or parthenogenetic activation, at the time when pronuclear formation occurs (Clayton, L., Stinchcombe, S.V. and Johnson, M.H., Zygote 1, 333–44, 1993). In order to investigate this developmental control of surface expression of uvomorulin, we examined the effects of inhibitors of various cellular processes on the appearance of uvomorulin at the oocyte surface, as assessed immunocytochemically. Inhibitors of cytoskeletal assembly (cytochalasin D and nocodazole), protein synthesis (puromycin and anisomycin), and DNA synthesis (aphidicolin) had no effect on surface expression. Brefeldin A, which inhibits intracellular transport and secretion, did prevent surface expression, but monensin did not. The effects of brefeldin were reversible; following 8 h of treatment, recovery of surface expression after removal of brefeldin began within 2 h. The time-course of surface expression post-activation suggested a link with pronuclear formation. However, when pronuclear formation was advanced experimentally using 6-dimethylaminopurine(DMAP), concomitant advancement of surface uvomorulin was not observed. Similarly, surface expression of uvomorulin did not accompany puromycin-induced pronuclear formation in maturing meiotic metaphase 1 (MI) oocytes in vitro. Thus, surface uvomorulin expression does not appear to be linked simply to pronuclear formation. Proteolytic processing of both newly synthesised and total uvomorulin to generate mature molecule from precursor increased within 30 min to 1 h after activation, and also occurred in the continued presence of brefeldin, suggesting that uvomorulin processing appears to be controlled independently of its suface expression.

The effect of iodoacetamide (IA), a sulphydryl blocking agent, on fertilisation in the mouse was examined by transferring zona-free oocytes into IA (500 μM)-containing medium at various times after insemination. When inseminated oocytes were transferred into IA medium 10 min after insemination, the oocyte chromosomes remained aggregated in one or two masses and the sperm nucleus failed to decondense. When oocytes were transferred during the second meiosis, oocytes meiosis was more or less arrested. The sperm nucleus decondensed but remained blocked at an early stage of decondensation. These observations suggest that thiol groups in the oocyte's cytoplasm and perhaps microtubules of the meiotic spindle play crucial roles in the completion of meiosis and the transformation of sperm nucleus into pronucleus.

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.

Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.