The objective of this study was to determine whether porcine PAG (poPAG) genes are expressed in embryos as they develop from the 1-cell stage to expanded blastocysts, and whether expression differed according to how embryos had been derived. Embryos at various preimplantation stages were assayed after in vivo fertilisation, after in vitro fertilisation of in vitro-matured oocytes, or following parthenogenetic activation of in vitro-matured oocytes. The presence of PAG transcripts was determined at the1-, 2-, and 4-cell, compact morula and blastocyst stages by reverse transcription-PCR procedures with PAG 1- and PAG 2-specific primers, followed by Southern blotting. The mRNAs for poPAG 1 and 2 were detected in in vitro-derived, in vivo-derived and parthenogenetically derived blastocyst stage embryos. In some replications poPAG 1 could be detected as early as the compact morula stage and poPAG 2 could be detected as early as the 4-cell stage. Our study revealed that poPAG 1 and 2 genes are expressed as early as the compact morula stage and 4-cell stage, respectively, in normal embryos and in parthenogenetically derived blastocysts. Thus it appears that the poPAGs are not maternally imprinted and they may be useful as potential candidates for markers of developmental competence.

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda–rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda–rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p>0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda–rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.

The present study investigates the role of catecholamines in the regulation of Bufo arenarum oocyte maturation. The metabolic changes in the oxidation of carbohydrates and the meiotic resumption evinced by the germinal vesicle breakdown were used as indicators of cytoplasmic and nuclear maturation, respectively. The results obtained suggest that noradrenaline (norepinephrine) could be one of the factors responsible for the metabolic behaviour that characterises cytoplasmically immature oocytes. The use of adrenaline (epinephrine), on the other hand, induced a metabolic change which made oocytes cytoplasmically mature. The effect of both catecholamines, which was dose-dependent, was observed in ovarian oocytes (surrounded by follicle cells) as well as in coelomic oocytes (free from follicle cells), suggesting the presence of adrenergic receptors in the gamete. The results obtained using adrenergic agonists and antagonists suggest that the effect of adrenaline would be due to an interaction with β2-receptors. Although catecholamines have an influence on the determination of the stage of cytoplasmic maturation of the oocytes, they do not affect nuclear maturation by themselves. Nevertheless, pretreatment of follicles with adrenaline caused a significant inhibition in progesterone-induced nuclear maturation even though this effect was markedly weaker when using noradrenaline.

Pig oocytes at metaphase II were activated by penetration of spermatozoa in cycloheximide-free and cycloheximide-containing fertilisation media. The precise nuclear stage, and the kinetics of degradation of cyclin B1 and dephosphorylation of MAP kinase were assessed after insemination. After maturation culture, 96% of oocytes reached metaphase II. At 6 h after insemination in cycloheximide-free medium, 68% of the oocytes were activated and had progressed to anaphase II or beyond. After 8 h, 89% of the oocytes were activated: a female pronucleus had formed and the heads of penetrating spermatozoa had enlarged and changed to male pronuclei. In the cycloheximide-containing medium, activation of oocytes started earlier than in cycloheximide-free medium. After 4 h, 43% of the oocytes were activated, and the percentage increased to 97% after 6 h. Pig cyclin B1 disappeared in the oocytes at 6 h after insemination in both cycloheximide-containing and cycloheximide-free media. Pig oocytes at metaphase II contained two types of MAP kinase – ERK 1 and ERK 2 – in their active phosphorylated forms. At 8 h after insemination ERK 2 changed to the fast-migrating inactive form in the oocytes cultured in both cycloheximide-containing and cycloheximide-free media, although the shift-down was not complete. The change was delayed by 2 h after the degradation of cyclin B1 molecules. These results demonstrate that degradation of pig cyclin B1 molecules corresponds to the transition of the oocytes from metaphase II arrest to anaphase II/telophase II and was followed by MAP kinase dephosphorylation.

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm–oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17–19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P<0.005) and uninjected control oocytes (5/84, P<0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.

In the current widely used round spermatid injection (ROSI) protocol for the mouse, the spermatid nucleus is separated from most of the cytoplasm before ROSI by drawing a spermatid in and out of a pipette. This results in the highest rate of normal fertilization. However, this separation method is not always consistent and can be time-consuming. An alternative separation method that cuts away the cytoplasm using the tip of an injection pipette was developed. After removing the cytoplasm, ROSI was performed following both post- and pre-activation protocols and development in vitro and in vivo were examined. The new method consistently removed the bulk of the cytoplasm, as shown by quantifying mitochondria. ROSI without the cytoplasm resulted in significantly higher rates of fertilization than ROSI with the cytoplasm into either post- or pre-activated oocytes. Furthermore, the offspring production rates of ROSI without the cytoplasm were also high (50% and 49% for the post- and pre-activation protocols, respectively). This new method for separating the cytoplasm is an alternative way of producing offspring using ROSI.

The migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the ‘spermatozoidal activator factor’ (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.

There is very little information available on stage-specific gene expression during early embryo development, particularly in the pig. Here, we accurately identified the genes that are specifically or prominently expressed in parthenogenetic porcine blastocysts as compared with 2-cell stage embryos. We accomplished this by using a PCR technology regulated by annealing control primers (ACPs). By utilizing 120 ACPs, a total of 46 expressed sequence tags (ESTs) of genes that are differentially expressed in blastocysts as compared with 2-cell stage embryos were cloned and sequenced. The cloned genes or ESTs all exhibited significant sequence similarity with known genes or ESTs of other species. Of the known genes, six genes [renin-binding protein (RNBP), BMDP, solute carrier family 25 (SLC25A6), MTHFD1, TRK-fused gene (TFG), spermidine synthase (SRM)] were selected and their stage-specific expression levels in porcine parthenotes were determined by real-time quantitative polymerase chain reaction at the 1-, 2-, 4-cell, morula and blastocyst stages. While RNBP, BMDP, SLC25A6, MTGFD1 and SRM were highly expressed only at the blastocyst stage, TFG was highly expressed at the 1-cell stage, then declined after genomic activation, high levels of expression being again detected at the morula and blastocyst stages. This analysis suggests that the ACP system is an effective tool for use in the identification of stage-specific genes in small numbers of porcine parthenotes. Examination of the genes differentially expressed in the blastocyst, which we have identified here, will provide insight into the molecular basis of preimplantation development.

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus–oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

In a previous study of mouse tetraploid[harr ]diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in postimplantation stage tetraploid[harr ]diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid[harr ]diploid chimaeric blastocysts.

The process of oocyte maturation is underlined by a redistribution of cellular organelles, among which mitochondria play a functional role for the acquisition of fertilization and developmental competence. In this paper, we applied electron and confocal microscopy by using DIOC6 and JC-1 stain to evaluate mitochondria distribution pattern and activity during different stages of oocyte growth in the ascidian Styela plicata. Three categories of oocytes at the germinal vesicle stage underlying the vitellogenic process were characterized on the basis of size, pigmentation and accessory cells. Mitochondria were spread throughout the cytoplasm at the smallest oocyte stage and gradually migrated to the periphery of the subcortical cytoplasm at the intermediate stage. At the fully grown oocyte stage, mitochondria were aggregated in the subcortical cytoplasm. This pattern of polarized mitochondria distribution correlates significantly with an increase in mitochondria potential and activity. In this paper we discuss the relationship of mitochondria to the acquisition of oocyte developmental competence.

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus–cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey–rabbit embryo). The monkey–rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey–rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey–rabbit ISCNT and rabbit–rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey–rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit–rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey–rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey–rabbit ISCNT embryos is similar to that of rabbit–rabbit SCNT embryos; and (4) the mitochondrial fate of monkey–rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

In this study we analyzed whether the in vivo storage of oocytes (time after ovulation until fertilization) affects the survival and the ploidy status of the yellowtail tetra Astyanax altiparanae. Fish were induced to spawn and, after ovulation, a small aliquot was stripped and immediately fertilized (positive control group). Subsequently, aliquots (~150 oocytes) were stripped and fertilized at various time points of 60, 120, 180 or 240 min. Developmental stages, abnormalities, survival and the ploidy status of the hatched larvae were examined. As expected, in the control group, 100% of the larvae were diploid. Conversely, triploid individuals were observed just at the 60 min treatment time point (0.6%). In vivo storage of oocytes also influenced the survival rates (P < 0.05); the 180 and 240 min samples, respectively, presented lower survival rates at gastrula (50.10±6.26% and 40.92±5.32%), and somite (17.80±5.14% and 4.41±2.76%) stages and lower hatching rates (12.01±4.04% and 4.41±2.76%). A higher percentage (99.27±0.40%) of normal larvae and only a few abnormal larvae (0.73±0.40%) were observed in the control group (P = 0.0000). This observation did not differ from that observed at the 60 min treatment point (P = 0.9976). A significant increase in the percentage of abnormalities was observed in the other treatments, and, after 240 min, the highest percentage of abnormal larvae was seen (P=0.0024; 83.33±16.67%). In conclusion, we showed that oocyte ageing had a significant effect on survival and may affect the ploidy status in A. atiparanae.

Clinical outcomes following frozen–thawed cleavage embryo transfer versus frozen–thawed blastocyst transfer in high responder patients undergoing in vitro fertilisation/intracytoplasmic sperm injection cycles are still debated. In a retrospective study, 106 high responder patients who were candidate for ‘freeze-all embryos’ were recruited. Frozen–thawed embryos were transferred at the cleavage stage (n = 53) or the blastocyst stage (n = 53). Clinical pregnancy was considered as the primary outcome and chemical pregnancy, ongoing pregnancy, implantation rate, and fertilization rate, as well as miscarriage rate, were measured as the secondary outcome. Clinical (47.2% vs. 24.5%), chemical (56.6% vs. 32.1%), and ongoing pregnancy rates (37.7% vs. 17%) as well as implantation rates (33.6% vs. 13.5%) were significantly higher in the blastocyst group compared with the cleavage group respectively (P < 0.05). Miscarriage rate was comparable between groups (P > 0.05). Transfer of frozen–thawed embryos at the blastocyst stage was preferable in the high responder patients to increase implantation, pregnancy and live birth rates compared with cleavage stage embryo transfer.

In this study we investigated ovulation in vitro using ovary samples from Bufo arenarum with respect to their response to stimulation with homologous pituitary homogenate (HPH) or with progesterone and prostaglandins (PGF2α and PGE1) as intermediates of pituitary action. Ovary samples were obtained from animals captured during the breeding period. Our results demonstrate that the ovulatory response to all different inducers was dose dependent, the highest percentage of ovulated oocytes being obtained with HPH treatment. An important increase in the ovulatory response was obtained by the association of PGF2α with either HPH or progesterone at suboptimal doses, indicating that this prostaglandin induced a synergistic potentiating effect. Incubation with cyclooxygenase inhibitors (indomethacin or diclofenac sodium) produced a significant decrease in the ovulation induced by HPH, demonstrating that prostaglandins are involved in the action of the pituitary gland in this process. According to our results, PGE1 not only had no participation in the ovulatory process, but also produced an inhibitory effect on ovulation induced by HPH treatment.

The aim of this study was to evaluate the advantages of the two-step embryo transfer (ET) strategy combining a day 2/3 ET with a day 5/6 blastocyst transfer. In an observational comparative study, 400 infertile women were enrolled from two assisted reproductive technology (ART) units according to inclusion criteria: age below 42 years and at least three embryos obtained on day 2 thus allowing an extended in vitro culture. Two groups were defined according to the ET strategy adopted: group 1 had a two-step ET; and group 2 had a day 2/3 ET with (subgroup 2a) or without (subgroup 2b) blastocysts cryopreserved on day 5/6. Live birth rate was significantly higher in group 1 than in subgroups 2a and 2b (36.5% versus 29.4% and 13.4%, respectively; p < 10−3). Multiple pregnancy rates were comparable between groups. After adjusting on major prognostic factors, the two-step ET strategy was still associated with a significantly higher live birth rate than the day 2/3 ET (OR = 2.23; 95% CI: 1.32–3.77). The two-step ET provides better live birth rates than the cleavage-stage ET. It does not increase multiple pregnancy rates if the number of embryos transferred is limited. It also prevents cycle loss when embryos fail to develop into blastocysts.

In this study, we investigated the effects of methyl-β-cyclodextrin (MBCD) on in vitro fertilization and the subsequent development of bovine oocytes. Bovine oocytes matured in serum-free medium were inseminated with frozen–thawed sperm pre-incubated in protein-free modified Brackett and Oliphant medium (BO) containing various concentrations of MBCD for various periods. MBCD decreased the frequency of live sperm, however enhanced the capacitation and acrosome reaction of the live sperm. Pre-incubation of sperm with 0.5, 1.0 and 1.5 mM MBCD for 2 and 4 h increased the frequency of normal fertilization. Embryos derived from oocytes fertilized with spermatozoa pre-incubated with MBCD developed normally to the blastocyst stage and term. There were individual differences and similar tendencies in four different sires in terms of the effects of MBCD upon fertilization. These results indicate that the pre-incubation of bovine sperm with MBCD affects viability and capacitation status of the sperm and promotes fertilization in vitro. Embryos derived from oocytes fertilized with sperm pre-incubated with MBCD developed normally to the blastocyst stage and term.

Local application of the agonists of serotonin receptors of third type 5-HTQ, SR57277A and quipazine into the interblastomere cleft of the sea urchin Paracentrotus lividus embryo during first cleavage division, evokes specific membrane currents, whereas application of these drugs out of contact area show currents of lower amplitude and longer latent period. At the same time 5-HT3-receptor agonist quipazine imitates interblastomere signal in half embryos, but corresponding antagonists prevent it. Present data develop the hypothesis of protosynapse, demonstrating that the distribution of membrane serotonin receptors is limited to the period of cleavage division and localized in the interblastomere contact area. A possible role of spatial–temporal restriction of receptors at the interblastomere contact area is discussed in relation to the subsequent embryo development.