We report that the first localized Ca2+ transient visualized in the blastodisc cortex of post-mitotic zebrafish zygotes has unique features. We confirm that this initial ‘furrow positioning’ Ca2+ transient precedes the physical appearance of the first cleavage furrow at the blastodisc surface and that it has unique dynamics, which distinguish it from the subsequent furrow propagation transients that develop from it. This initial transient displays a distinct rising phase that peaks prior to the initiation of the two linear, subsurface, self-propagating Ca2+ waves that constitute the subsequent furrow propagation transient. Through the carefully timed introduction of the Ca2+ buffer, dibromo-BAPTA, we also demonstrate the absolute requirement of this initial rising phase Ca2+ transient in positioning the furrow at the blastodisc surface: no rising phase transient, no cleavage furrow. Likewise, the introduction of the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-aminoethoxydiphenyl borate, eliminates both the rising phase transient and the appearance of the furrow at the cell surface. On the other hand, antagonists of the ryanodine receptor and NAADP-sensitive channels, or simply bathing the zygote in Ca2+-free medium, have no effect on the generation of the rising phase positioning transient or the appearance of the furrow at the surface. This suggests that like the subsequent propagation and deepening/zipping Ca2+ transients, the rising phase furrow positioning transient is also generated specifically by Ca2+ released via IP3Rs. We propose, however, that despite being generated by a similar Ca2+ release mechanism, the unique features of this initial transient suggest that it might be a distinct signal with a specific function associated with positioning the cleavage furrow at the blastodisc surface.

Exposure to lead may cause changes in the male reproductive system. We evaluated the effect of lead chloride (PbCl2) in vitro on semen quality from 31 individuals. Samples were incubated at room temperature for two exposure times (4 h and 8 h) and with two concentrations of PbCl2 (15 μg/ml or 30 μg/ml). Results showed that PbCl2 significantly inhibited rapid progressive motility and caused an increase in the percentage of tail anomalies in both times and concentrations assessed, as well as a decrease in vitality in the group exposed to 30 μg/ml PbCl2. A significant increase in immotile sperm was also observed between the group control and the groups submitted to lead. Total motility and DNA fragmentation also showed a significant decrease and increase, respectively, after 4 h of incubation in the group exposed to 30 μg/ml and in both groups after 8 h of incubation. In conclusion, PbCl2 affected sperm parameters and DNA integrity, which are essential for male fertility.

The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8–16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: ‘RNA processing’, ‘translation’ and ‘mRNA metabolic process’. Genes that are important to the molecular functions of ‘RNA binding’ and ‘translation factor activity, RNA binding’ were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

We have investigated the fertilisation competence, protein synthesis, histone H1 kinase and myelin basic protein (MBP) kinase activities in three categories of bovine oocytes (derived from three size categories of follicles: M–medium, 2.5–5.0 mm; S–small, 1.5–2.5 mm; T – tiny, 1.0–1.5mm). In contrast to more or less normal meiotic maturation (85.6%) and fertilisation (70.8%) of M oocytes cultured for 24h, the fertilisation of M oocytes cultured for 40h was associated with increased rates of retarded male pronuclear development and retention of the second polar body. The S and T oocytes cultured for 24h or 40h were mostly arrested at defective late diakinesis - metaphase I (77.5–100%) stage. After fertilisation of S and T oocytes cultured for 24h no polar body was extruded and formation of one, three or four female pronuclei, together with mostly normal male pronuclei, was observed. The fertilisation of S and T oocytes after 40h culture resulted in a higher number of female and a decreased number of male pronuclei. A major change in the pattern of protein synthesis was associated with the resumption of meiosis. There were no significant differences in the profile of protein synthesis between oocyte categories in all groups either matured or fertilised. The H1 kinase activity reached comparable increased levels in oocytes of all categories matured for 24h and decreased during the 40h culture, most significantly in M oocytes. The MBP kinase activity was at approximately the same high level in all categories of oocytes after 24h of culture and remained stable until 40h. The fertilisation after 24h of culture resulted, in M oocytes, in low levels of both H1 and MBP kinase activities; in S oocytes, only H1 kinase was completely inactivated while MBP kinase activity decreased to some extent; in T oocytes, both H1 and MBP kinase activity decreased. Fertilisation of all oocyte categories after 40h culture resulted in complete inactivation of both these kinases to their basal levels.

We have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The ‘perivitelline’ block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.

The low involvement of glucose metabolism in early preimplantation embryos has suggested the presence of metabolic blocks in the glycolytic pathway. Genetic expression of hexokinase (HK), glucose-6-phosphate isomerase (GPI) or phosphofructokinase (PFK) were qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in individual human immature oocytes at the germinal vesicle (GV) stage and in single non-fertilised human metaphase II (MII) oocytes, after IVF or ICSI failures. Transcripts encoding for HK, GPI and PFK were detectable in the majority of the oocytes analysed, and with different expression patterns. GPI transcripts were consistently present and appear to be constitutively expressed during GV and MII stages. In contrast, low quantities of transcripts encoding for HK and PFK were observed in all oocytes analysed. Our data revealed that the metabolic block previously reported at GPI or PFK levels underwent post-transcriptional regulation. The expression profiles of HK and PFK transcripts in GV and MII reflect a low level of transcription or active translation of maternal transcripts during oocyte maturation. Nevertheless, enzymatic activities of HK and PFK have previously been determined in human oocytes. This suggests that HK and PFK may be accumulated in protein (enzyme) form instead of maternal mRNA during human oocyte maturation. The expression pathway of glycolytic metabolism reflects the presence of different mechanisms involved in gene expression/regulation at the transcriptional and translational level and their accumulation during human oocyte maturation.

We performed fertilization experiments with Acropora digitifera, which is one of the dominant scleractinian corals in the Ryukyu Archipelago, Japan, to determine optimal conditions for in vitro manipulations. Our result suggests that conspecific fertilization is essentially complete within 30 min under the experimental conditions used in usual fertilization experiments in corals. Previous in vitro experiments (1 × 105–106 sperm/ml, 4–8 h) are likely to have overestimated the efficiency of fertilization of Acropora spp. in the field. Therefore, we suggest that incubation periods shorter than those used to date (i.e. complete exclusion of sperm 1 h after their addition) would be more appropriate for the estimation of fertilization rates in corals.

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.

Spermatogenesis is a dynamic process that culminates in the production of mature spermatozoa in the seminiferous tubules of sexually mature animals. Although sperm leaving the testis are fully differentiated, they must further undergo two additional maturation steps before acquiring the capability to fertilize the egg. Such processes take place during the epididymal residency and transport in the seminal fluid during ejaculation and, after delivery into the female reproductive tract, during the journey aiming the encountering the egg in the oviduct. Throughout this trip, spermatozoa are exposed to different reproductive fluids whose molecular compositions regulate the progress towards obtaining a fertilized competent cell. This review summarizes the evidence obtained so far supporting the participation of male and female reproductive tract-derived proteins in the modulation of sperm fertilizing ability and discusses the mechanisms by which such regulation may be accomplished.

After spermatozoa bind to and penetrate the extracellular matrix of the egg, the zona pellucida, they adhere to and fuse with the plasma membrane of the egg. Since sperm–egg fusion may involve membrane glycoproteins and/or carbohydrate binding proteins, we sought to test this hypothesis by challenging sperm–egg fusion in hamster and in mouse with added carbohydrates. In this study, a number of carbohydrate and glycoconjugates were examined for their ability to inhibit sperm–eggfusion. In the hamster, D(+)-glucosamine, D(+)-galactosamine, albumin-bovine-glucosamide and-galactosamide, fucoidan and dextran sulphate inhibited the fusion of spermatozoa with zona-free eggs. The same effects were seen in the mouse, except for the toxic effects of D(+)-galactosamine. These facts suggest a role of carbohydrate binding proteins or glycoproteins in the fertilisation process at the level of binding to and fusing with the oolemma.

Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles.

The present study investigated the effects of the sexual maturity of oocyte donors on in vitro maturation (IVM) and the parthenogenetic developmental capacity of fresh minke whale oocytes. The effects of cytochalasin B (CB) pretreatment and two types of cryoprotectant solutions (ethylene glycol (EG) or ethylene glycol and dimethylsulfoxide (EG + DMSO)) on the in vitro maturation of vitrified immature whale oocytes were compared, and the developmental capacity of vitrified immature whale oocytes following IVM and intracytoplasmic sperm injection examined (ICSI). The maturation rate did not differ significantly with sexual maturity (adult, 60.9%; prepubertal, 53.1%), but the parthenogenetic activation rate of oocytes from adult donors (76.7%) was significantly higher (p < 0.05) than that of oocytes from prepubertal donors (46.4%). The maturation rates after vitrification and warming were not significantly different between the EG (22.2%) and EG + DMSO groups (30.2%), or between the CB-treated (30.4%) and non-CB-treated groups (27.3%). These results indicate that parthenogenetic activation of in vitro matured oocytes from adult minke whales was superior to that from prepubertal whales, but that the developmental capacity of the whale oocytes after parthenogenetic activation or ICSI was still low. The present study also showed that CB treatment before vitrification and two kinds of cryoprotectants did not improve the IVM rate following the vitrification of immature whale oocytes.

It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 μg/ml estradiol-17β. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6–7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes.

We report evidence to suggest that during the first few meroblastic cell divisions in zebrafish embryos a dynamic population of central-spindle microtubules serve a crucial function in positioning the cleavage furrow at the surface of the blastoderm. Originating from the mid-zone of the mitotic spindle they develop into what we term a mid-spindle ‘pre-furrowing microtubule array’ that expands upward and outward from the spindle mid-zone towards the blastodisc surface. We suggest that this structure transmits positional information to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. We also propose that the pre-furrowing microtubule array then develops into a furrow-ingression microtubule array that helps direct and assemble the deepening furrow as it cuts its way through the blastodisc. Due to the location of its origin, the pre-furrowing microtubule array serves to successfully separate the daughter nuclei and thus equally divide the blastoderm. Furthermore, co-localization with elements of the cortical endoplasmic reticulum and their inositol 1,4,5-trisphosphate receptors suggests that the pre-furrowing microtubule array may also play a role in organizing localized Ca2+ transients that have been shown to be essential to the furrow positioning, propagation and deepening process during cytokinesis in zebrafish embryos.

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.

Mutation in CFAP43 leads to severe asthenozoospermia and multiple morphological abnormalities of the sperm flagellum (MMAF) in both human and mouse. Previous studies have shown that disruption of intra-manchette transport (IMT) caused failure of flagellum assembly and sperm head shaping. In a previous study, therefore, we postulated that disruption of IMT may contribute to the failure of sperm flagellum formation and result in MMAF, however the mechanisms underlying these defects are still poorly understood. Cfap43-deficient mice were studied here to reveal the cellular mechanisms of abnormal sperm head morphology and MMAF. Depletion of Cfap43 led to abnormal spermiogenesis and caused MMAF, sperm head abnormality and oligozoospermia. Furthermore, both abnormal manchette and disorganized ectoplasmic specialization (ES) could be observed at the elongated spermatids in Cfap43-deficient mice. Therefore, our findings demonstrated that, in mice, CFAP43-mediated IMT is essential for sperm head shaping and sperm flagellum formation.

To evaluate the integrity of genomic imprinting in embryos that failed to develop normally following intracytoplasmic sperm injection (ICSI), we analysed the methylation profile of H19 and KCNQ1OT1 imprinting control regions, H19DMR and KvDMR1 respectively, in high-grade blastocysts and in embryos that exhibited developmental anomalies. Significant hypomethylation of KvDMR1 was specifically observed in 5/5 atypical blastocysts graded BC, which probably reflected the vulnerability of the imprint in the inner cell mass during the methylation remodelling phase in the early embryo. In addition, KvDMR1 was hypermethylated in 2/5 CC graded atypical blastocysts and in 2/8 embryos that exhibited developmental delay. H19DMR appeared differentially methylated in all groups of embryos. DNA methyltransfersase 1 (DNMT1) expression was similar in most of the tested embryos and could not account for the abnormal methylation patterns of KvDMR1 observed.

Alcohol is known to preserve genomic DNA and the primary structure of sperm protamines. To determine whether alcohol can retain the genetic and reproductive potential of mammalian sperm nuclei, mature mouse spermatozoa were stored in 70% ethanol or propanol for up to 2 months before injection into oocytes. Live offspring were obtained after injection of spermatozoa stored in 70% ethanol for 1 day at -20 °C. About 20% of the spermatozoa stored under this condition had normal chromosomes. The remaining 80% of spermatozoa and all the spermatozoa stored in 70% ethanol for 2 months had structurally aberrant chromosomes, and none could support the development of normal embryos. High concentrations of alcohol do not alter the primary structure of either DNA or small-molecular-weight protamines. However, alcohol may modify protamine—protamine or protamine—DNA interactions in a manner that results in the induction of DNA strand breaks during sperm chromatin decondensation within the oocyte. The limited success in obtaining normal offspring with ethanol-stored spermatozoa is encouraging. It may be possible to overcome these problems and develop a simple method for preserving mammalian spermatozoa without freezing.

This study shows for the first time the presence of a jelly coat on oocytes of neotropical Characiformes fish. This structure could be responsible for the adhesiveness of Astyanax bimaculatus oocytes, a species widely distributed in South America including in the São Francisco River basin in Brazil. Adult specimens of A. bimaculatus were submitted to artificial reproduction in order to analyse the egg morphology and embryonic development. The eggs were fertilised and kept in incubators with a water temperature of 24°C so that embryogenesis could be monitored. Ovulated and unfertilised oocytes were also collected and submitted to routine histological techniques. Astyanax bimaculatus oocytes were found to be spherical, yellowish, and covered by a thin jelly coat with a slightly adhesive surface. The mean oocyte diameter was 1.03 ± 0.03 mm, the perivitelline space was 0.21 ± 0.02 mm and the jelly coat's thickness was 0.04 ± 0.01 mm. Positive periodic acid–Schiff (PAS) stain and Alcian blue stain pH 2.5 indicated the presence of neutral glycoproteins, and carboxylated acid glycoconjugates on the jelly coat that formed mucosubstances that may be associated with egg adhesiveness. At a water temperature of 24°C, blastopore closure and hatching occurred at 5 h and 17 h after fertilisation, respectively. The results of this study provide essential information for phylogenetic studies and for a better understanding of the reproductive strategy of A. bimaculatus, currently included in the incertae sedis group of the Characidae family due to the lack of monophyly among the families of the group.

Oviductal oocytes (n = 795) were obtained from ovulation-induced prepubertal gilts. In the first experiment, different parameters related to in vitro fertilisation (IVF) were compared in the presence and absence of cumulus matrix (which is shed with the oocytes at ovulation.) The results show that the presence of this matrix is beneficial because the rates of fertilisation (69%) (number of monospermic oocytes/total number of healthy mature oocytes; 42%), and the median number of spermatozoa per oocyte (1.52±0.06), were improved with respect to those obtained in its absence (54%, 22% and 2.33 ± 0.08, respectively; p<0.01). In the second experiment the effect of two different volumes of co-incubation medium (2 ml and 0.4 ml) on the same parameters of porcine IVF were compared. No significant differences between volumes were observed, except in the mean number of spermtozoa per oocyte and the percentage of dispermic oocytes.