The tolerance of the oocyte plasma membrane (oolemma) to electrical pulses (TEP) was investigated for oocytes, zygotes, and embryos at the early and late 2-cell stage. The oocyte survival rate after two electrical pulses (1.4KV/cm for 640μs each) was used as an indicator of the TEP of the oolemma. Survival rate of mid-pronuclear zygotes (94.3%±2.3%) was significantly (p < 0.05) higher than that of recently ovulated (2.1%±1.9%) and in vivo aged (25.1%±2.6%) oocytes; survival rate of in vivo aged oocytes was also significantly higher than that of recently ovulated oocytes. Soon after fertilisation, the survival rate of the oocytes markedly increased, up to 94% at the mid-pronuclear stage. Survival ratedropped thereafter. These results suggest that the characteristics of the oocyte plasma membrane (oolemma) change after fertilisation.

The c-fos and c-jun proto-oncogenes are involved in the regulation of gene expression, cell proliferation, differentiation and tumorigenesis. Embryogenesis, like tumorigenesis, involves dramatic cell growth, cleavage and differentiation processes. Activation of the c-fos and c-jun proto-oncogenes in sheep conceptuses during the period of rapid growth and elongation was examined using reverse transcription and polymerase chain reaction (RT-PCR). The specificity of PCR products was determined by Southern blot hybridisation analysis with a non-radioactive DNA probe. A band corresponding to a 507 bp fragment (predicted amplified c-fos gene cDNA product) was observed in 3 of 9 day-13, 1 of 4 day-14 and 1 of 2 day-16 embryos. Meanwhile, a 400 bp c-jun transcript was aslo detected in 1 or 2 day-12, 3 of 9 day-13 and 2 of 2 day-16 embroyos. These results suggest that mRNA transcripts of c-fos and c-jun proto-oncogenes were specifically expressed during the period of ovine embryonic elongation and may have a possible role in preimplantation embryonic development of sheep.

Mouse oocytes penetrated by spermatozoa during germinal vesicle (GV) breakdown undergo maturation and are arrested at metaphase of the second meiotic division despite the presence of sperm nuclei within the ooplasm. When these oocytes were re-inseminated, none was penetrated by newly added spermatozoa. When GV oocytes were inseminated and cultured in the presence of dibutyryl cAMP, the oocytes remained at GV stage, yet they did not permit entry of additional spermatozoa. These observations suggest that the plasma membrane of maturing oocytes is modified by precociously penetrating spermatozoa independently from cortical granule exocytosis. Sperm components incorporated into the oocytes seem to be responsible for the modification of the oocyte's plasma membrane.

Following fertilisation, the sperm triggers a series of intracellular changes which initiate oocyte activation and pronuclear formation. Oocyte activation can also be induced artificially by several chemicals, such as the calcium ionophore A23187. The sperm nucleus is transformed into the male pronucleus through the interaction of oocyte cytoplasmic factors. The profile of protein synthesis is different in bovine oocytes following fertilisation and parthenogenetic activation. The formation of male and female pronuclei was not blocked by the presence of the protein synthesis inhibitor cycloheximide. These results suggest that bovine oocyte activation by sperm and parthenogenetic activation induce different cytoplasmic responses for protein synthesis and that new protein synthesis is not required for male pronuclear formation in bovine zygotes.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

The presence of RNase A activity was studied in vitro in homogenates of quail oocytes and early embryos using [3H]poly(U) as a substrate. The activity was measured by adsorption of the undegraded substrate onto DE-81 filter paper discs and by chromatographic separation on a Sephadex G-50 column. RNase A activity examined by these methods was almost undetectable in quail previtellogenic, vitellogenic and ovulated oocytes as well as in the embryos from laid eggs. It is estimated to be about 1.1 × 10−5 Kunitz units per ovulated oocyte. Higher activity starts to appear in gastrulating embryos. These findings are discussed in relation to other studies demonstrating the high stability of maternal RNA during early development, especially in growing oocytes.

Hypoxanthine can block preimplantation mouse embryo development in vitro at the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the hypoxanthine-induced block. While IBMX alone is unable to reverse the hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Previous studies have shown that mouse sperm bind to hamster eggs and hamster sperm bind to mouse eggs in vitro. Furthermore, sperm receptor glycoprotein isolated from the zona pellucida of unfertilised hamster (hZP3) and mouse (mZP3) eggs binds to sperm from the heterologous species. Here, we expressed the hZP3 gene, under control of a constitutive promoter (pgk-1), in mouse embryonal carcinoma (EC) cells and Chinese hamster ovary (CHO) cells stably transfected with the hZP3 gene. In both cases, recombinant hZP3 (EC-hZP3 and CHO-hZP3) secreted into the culture medium was partially purified by high-performance liquid chromatography on a size-exclusion column and assayed for bioactivity using mouse and hamster gametes. Unlike hamster egg hZP3, which binds to both mouse and hamster sperm, EC-hZP3 and CHO-hZP3 exhibits species-specific binding to hamster sperm and induce hamster sperm, but not mouse sperm, to undergo the acrosome reaction in vitro. These results provide further evidence that species-specific binding of sperm to eggs in mammals is carbohydrate-mediated. Furthermore, the results suggest that recombinant forms of mammalian sperm receptors may be useful in assessing the molecular basis of species-specific fertilisation in mammals.

Parthenogenetically activated mammalian oocytes have been used in the past decade as cytoplasts, in an attempt to support the development of nuclear transplant embryos. The present experiments were undertaken to study the DNA synthesis and the organisation of microtubules, nuclear envelope and chromatin during the first cell cycle of electrically activated porcine oocytes (parthenotes) matured in vitro by using immunocytochemistry and laser scanning confocal microscopy. The results showed that pronuclear-like (PN) formation began 4–5 h post-activation (hpa), whilst DNA synthesis as revealed by bromodeoxyuridine incorporation was initiated 5–6 hpa, with a maximum number of labelled oocytes (73%) around 11 hpa, and persisted in some parthenotes until 15–16 hpa. In the metaphase II (MII) oocytes, microtubules were detected only in the metaphase II spindle; no lamin A/C antigen was observed. Electrical DC pulses resulted in 91% of MII oocytes being activated and confocal microscopy Indicated that microtubules were assembled in the spindle first for the extrusion of a second polar body, and for the second time for division from one to two cells. Nuclear envelope, indicated by anti-lamin A/C stain, was formed around the time of PN formation and surrounded the nuclear chromatin of 1- and 2-cell parthenogenotea. These results demonstrate that the apparent normality in both DNA synthesis and dynamics of microtubules and nuclear envelope is involved with chromosomal organisation in the parthenotes. In addition, the use of electrically activated IVM oocytes for both nuclear transfer and parthenogenetic studies in pigs is discussed.

Previous studies of tetraploid↔diploid mouse chimaeras and mosaics have revealed that tetraploid cells do not contribute equally to all tissues of the conceptus. In this study we have shown that, within 30 h of aggregating cleavage stage embryos, tetraploid cells were non-randomly distributed among different tissues of the early blastocyst. They were preferentially allocated to the mural trophectoderm regardless of cell size at the time of aggregation. This early effect may underlie the restricted distribution of tetraploid cells at later stages. We have demonstrated for the first time that ploidy can influence the relative position of blastomeres in the preimplantation embryo.

In this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.

The experimental objective was to determine whether the capability of bovine oocyte plasma membrane to bind sperm changes during in vitro oocyte maturation and fertilisation. Binding was quantified by the intensity of tetramethylrhodamine isothiocyanate (TRITC) fluorescence at the periphery of oocytes following incubation with biotinylated sperm plasma membrane proteins and subsequent incubation with TRITC-avidin. Bovine oocytes were matured in vitro. Sample groups were removed after 0,6 and 22 h, or inseminated and further cultured for 24 or 48 h. Oocytes were denuded of cumulus cells and zona pellucida and co-incubated with 56 μg biotinylated bovine sperm plasma membrane protein for 45 min in 150 μl drops of saline-BSA. Controls were incubated for the same time period in the absence of sperm plasma membrane proteins. All oocytes were rinsed, incubated with TRITC-avidin and subsequently fixed and transferred to mounting medium. Oocytes were scanned with a confocal microscope and analysed using ImageQuant software. The binding of sperm plasma membrane was quantified by integrated fluorescent intensity in standardised ellipses spaced around the plasma membrane of the oocyte. Values are expressed as mean intensity units per 320 pixel ellipse. Binding of sperm plasma membrane continued to increase throughout in vitro oocyte maturation and fertilisation (9051, 24318 and 49953 for 0 and 22 h in vitro matured oocytes and fertilised oocytes, respectively; p = 0.0001). A dramatic decrease in sperm plasma membrane binding to the oocyte plasma membrane was observed in 2-cell embryos (mean intensity = 24477, p = 0.0001). The observed binding was primarily due to the binding of sperm plasma membrane proteins, as control oocytes incubated with TRITC- avidin only were barely visible (integrated fluorescence intensity values ranged from 8 to 3757).

Jelly coat of starfish eggs has the capacity to activate homologous spermatozoa and induce the acrosome reaction. We have isolated 12 sperm-activating peptides (SAPs) from the egg jelly of the starfish, Asterias amurensis. Eleven SAPs were structurally identified by sequence analysis and electro-spray ionisation mass spectrometry. All of them are glutamine-rich tetratriacontapeptides with an intramolecular disulphide linkage between Cys8 and Cys32. They are much larger than sea urchin SAPs and do not show any significant sequence similarities to known proteins. Thus we have collectively named them asterosaps. The amino terminal region, where structural diversity of asterosaps is observed, is not important for their activity, whereas the disulphide linkage is essential. Asterosaps do not induce the acrosome reaction by themselves, but are able to induce the acrosome reaction in combination with an egg jelly glycoconjugate named ARIS. Furthermore, anti-asterosap rabbit antibody significantly decreased the acrosome reaction-inducing activity of the jelly solution and the activity was restored by addition of excess asterosap. These results support our hypothesis that the main physiological role of SAPs is the induction of the acrosome reaction in cooperation with two other jelly components, ARIS and Co-ARIS.

Prophase-arrested oocytes of Ruditapes philippinarum cannot be fertilised or stimulated by excess KCl, in contrast to the situation found in other bivalve species such as Barnea and Spisula. However, these oocytes can be triggered to reinitiate meiosis following treatment by serotonin or several pharmacological agents (calcium ionophores, thapsigargin, weak bases) which promote an intracellular calcium surge. Ruditapes oocytes further arrest in metaphase I, at which stage they can be activated either by sperm or by excess KCl. This suggests that functional voltage-operated calcium channels are expressed in this species during the course of maturation. Using pharmacological tools and direct binding of specific dihydropyridines, we demonstrate that these channels are L-type calcium channels which become functional after serotonin stimulation, their number increasing before germinal vesicle breakdown. Moreover we establish that: (1) the addition of 20 μM S(—)Bayκ8644, an agonist of L-type calcium channels, to metaphase-arrested oocytes releases them from metaphase block; (2) incubating these oocytes with PN200-110, a potent blocker of L-type calcium channels, inhibits their activation by both excess KCl and fertilisation. Taken together these data suggest that the absence of L-type calcium channels in the membrane of prophase-arrested oocytes of Ruditapes may account for their inability to be fertilised.

Nuclei of diplotene (dictyate) primordial oocytes (PO) were transferred to metaphase II oocytes and to activated mouse oocytes using cell fusion techniques. In a metaphase II oocyte, the PO nucleus condenses within 2–3 h to bivalents which become arranged on the first meiotic spindle. After oocyte activation, homologous chromosomes segregate between the oocyte and the first polar body, and a diploid pronucleus-like nucleus reforms from the one set of dyads. This nucleus condenses in the first embryonic mitosis into 40 ‘somatic’ chromosomes which coexist in the common metaphase plate with 20 somatic chromosomes originating from the female pronucleus. Shortening of the time between fusion and activation to about 1 h prevents bivalent differentiation. The PO nucleus condenses only partially and reforms, after oocyte activation, a pronucleus-like nucleus. This nucleus gives rise at the first embryonic mitosis to 20 bivalents which coexist with 20 somatic chromosomes originating from the female pronucleus. A PO nucleus introduced into an activated egg completes the first cell cycle as an intact interphase nucleus. It never condenses in the first embryonic mitosis into bivalents, and undergoes only initial condensation (preceding bivalent differentiation). These results indicate that: (1) condensation into bivalents, meiotic spindle formation and first meiotic division can be greatly accelerated by the introduction of an early diplotene (dictyate) oocyte nucleus into a metaphase II oocyte, and (2) depending on whether the diplotene nucleus enters the first embryonic (mitotic) cell cycle after just initiating or after completing the first meiosis, it gives rise at the first cleavage division to meiotic (bivalents) or ‘somatic’ chromosomes respectively.

This paper addresses the proposition, first advanced by Wilson (1925), that successful embryogenesis depends on an ordered series of events in oogenesis. It is at the completion of this varied set of intracellular changes that the oocyte finally acquires its full capacity to support fertilisation and development. Amongst the earliest nuclear events are those associated with chromosome pairing and meiotic recombination. During the growth phase cell volume increases 300-fold and the cytoplasm becomes the storage site for RNA and protein which will be mobilised during early development. Finally, a short phase of intracellular reprogramming, or maturation, completes the series of events during oogenesis that confer developmental competence upon the oocyte. Follicle cell support is an indispensable requirement for ordered oocyte development and provides the early germline cell with many of the essential nutrients and growth regulators required to ensure progression through the protracted growth phase (see contributions by Cecconi & Rosella and De Felici et al. this issue). Although different, the interactions between the full-grown oocyte and the antral follicle are no less crucial to the acquisition of competence than those involved in the earlier stages of oogenesis.

Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8h of initial culture) or metaphase I (12h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.