The aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL–1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

Sperm preparation is an important step in the in vitro production of embryos. Centrifugation through colloids has been used to select normal sperm for assisted reproduction in several species. Animal models can sometimes be used as a preliminary step to investigate sperm preparation methods that are potentially of use for human fertility treatments. In this study bovine semen was prepared using three variants of the single-layer centrifugation sperm selection technique (Small, Mini, Mini-EP) with Bovicoll (Androcoll-B). Computer-assisted sperm motility analysis, the hypo-osmotic swelling test, and the sperm chromatin structure assay were performed on unselected (control) and SLC-selected sperm samples. Mini and Mini-EP gave the highest yield of motile spermatozoa, progressive motility and membrane integrity. In vitro fertilization trials were performed to investigate the fertilizing ability of the frozen–thawed bovine spermatozoa selected with Bovicoll. Mini-SLC (single-layer centrifugation) and swim-up (Control) were performed and cleavage rate and blastocyst rate did not differ significantly between groups. As there was a trend to an increased number of cells in blastocysts in the SLC group, the Mini-SLC method is at least as good as swim-up for selecting frozen–thawed bull spermatozoa for in vitro fertilization (IVF). This method could potentially be used to prepare human sperm for assisted reproduction.

This work analyses the changes that caprine spermatozoa undergo during in vitro fertilization (IVF) of in vitro matured prepubertal goat oocytes and their relationship with IVF outcome, in order to obtain an effective model that allows prediction of in vitro fertility on the basis of semen assessment. The evolution of several sperm parameters (motility, viability and acrosomal integrity) during IVF and their relationship with three IVF outcome criteria (total penetration, normal penetration and cleavage rates) were studied in a total of 56 IVF replicates. Moderate correlation coefficients between some sperm parameters and IVF outcome were observed. In addition, stepwise multiple regression analyses were conducted that considered three grouping of sperm parameters as potential explanatory variables of the three IVF outcome criteria. The proportion of IVF outcome variation that can be explained by the fitted models ranged from 0.62 to 0.86, depending upon the trait analysed and the variables considered. Seven out of 32 sperm parameters were selected as partial covariates in at least one of the nine multiple regression models. Among these, progressive sperm motility assessed immediately after swim-up, the percentage of dead sperm with intact acrosome and the incidence of acrosome reaction both determined just before the gamete co-culture, and finally the proportion of viable spermatozoa at 17 h post-insemination were the most frequently selected sperm parameters. Nevertheless, the predictive ability of these models must be confirmed in a larger sample size experiment.

While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P<0.05) for T85A–eGFP-injected than for -uninjected or eGFP-injected embryos (73±7.3 vs. 28±7.3 or 14±6.7, respectively). We propose that ectopic expression of mutant AQPs may provide an avenue to improve cryopreservation results of large cells and tissues in which current vitrification protocols yield low survival.

The main aim of the present study was to examine the hypothesis that an increase in the number of granulosa cells surrounding developing bovine oocytes results in both high ATP levels and an increase in the acetylation level of H4K12 in oocytes grown in vitro. Oocyte–granulosa cell complexes (OGCs) were collected from early antral follicles (EAFs, 0.4–0.7 mm in diameter), and individually cultured on 96-well plates with or without additional granulosa cell mass that had been prepared from other OGCs. After 16 days of culture, we examined: (i) the rate of antrum formation of the OGCs; (ii) the diameter, maturation, and fertilization rate of the oocytes; and (iii) the ATP content and acetylation level of H4K12 in the oocytes grown in vitro. Granulosa cell mass added to the culture medium contributed to the development of OGCs with a higher rate of antrum formation and oocyte growth. Furthermore, the addition of granulosa cells increased the ATP content and acetylation level of H4K12 in oocytes grown in vitro compared with those developed without addition of granulosa cells. In addition, there was a positive correlation between the ATP content in oocytes grown in vitro and the number of granulosa cells in the corresponding OGCs. The results suggest that granulosa cells play a role not only in the development of OGCs and the growth of oocytes, but also in the determination of ATP content and the acetylation of H4K12 in the oocytes developed in vitro.

An important step for successful fertilization and further development is the increase in intracellular Ca2+ in the activated oocyte. It has been known that starfish oocytes become increasingly sensitive to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation to exhibit highly efficient IP3-induced Ca2+ release (IICR) by the time of germinal vesicle breakdown (GVBD). However, we noted that the peak level of intracellular Ca2+ increase after insemination is already high in the maturing oocytes before GVBD. Using maturing oocytes before GVBD, we investigated Ca2+ release mechanisms other than IICR. We report here that Ca2+-release mechanisms dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) and nicotinamide adenine dinucleotide (NADP), the precursor of NAADP, became functional prior to the development of IICR mechanisms. As with IP3, but unlike NAADP, the Ca2+ stores responsive to NADP are sensitized during the meiotic maturation induced by 1-methyladenine (1-MA). This suggests that the process may represent a physiological response to the maturation hormone. NADP-dependent Ca2+ release in immature oocytes, however, did not induce oocyte maturation by itself, but was enhanced by the conditions mimicking the increases of intracellular Ca2+ and pH that take place in the maturing oocytes of starfish.

The aim of this study was to investigate the influence of copper (Cu) during in vitro maturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu, P < 0.01) with respect to cells treated with Cu for cumulus–oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. After in vitro fertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.

In zebrafish embryos, distinct Ca2+ transients are localized to the early cleavage furrows during the first few cell division cycles. These transients are generated mainly by release via IP3Rs in the endoplasmic reticulum, and they are necessary for furrow positioning, propagation, deepening and apposition. We previously showed, via the use of inhibitors, that store-operated Ca2+ entry (SOCE) also appears to be essential for maintaining the IP3R-mediated elevated levels of [Ca2+]i for the extended periods required for the completion of successful furrow deepening and daughter cell apposition in these large embryonic cells. Here, newly fertilized, dechorionated embryos were fixed at various times during the first and second cell division cycles and immunolabelled with antibodies to STIM1 and/or Orai1 (key components of SOCE). We show that both of these proteins have a dynamic pattern of localization during cytokinesis of the first two cell division cycles. These new data help to support our previous inhibitor results, and provide additional evidence that SOCE contributes to the maintenance of the sustained elevated Ca2+ that is required for the successful completion of cytokinesis in the large cells of embryonic zebrafish.

The aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

The development of embryonic stem cells (ESCs) from large animal species has become an important model for therapeutic cloning using ESCs derived by somatic cell nuclear transfer (SCNT). However, poor embryo quality and blastocyst formation have been major limitations for derivation of cloned ESCs (ntESCs). In this study, we have tried to overcome these problems by treating these cells with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. First, cloned embryos were treated with Scriptaid to confirm the effect of HDACi on cloned embryo quality. The Scriptaid-treated blastocysts showed significantly higher total cell numbers (29.50 ± 2.10) than non-treated blastocysts (22.29 ± 1.50, P < 0.05). Next, cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free, reconstructed, four-cell-stage SCNT embryos were injected into the empty zona of hatched parthenogenetic (PA) blastocysts. Blastocyst formation and total cell number of cloned blastocysts increased significantly for all aggregates (76.4% and 83.18 ± 8.33) compared with non-aggregates (25.5% and 27.11 ± 1.67, P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine ES-like cell derivation. Aggregated blastocysts showed a higher primary colony formation rate than non-aggregated cloned blastocysts (17.6 ± 12.3% vs. 2.2 ± 1.35%, respectively, P < 0.05). In addition, derived ES-like cells showed typical characters of ESCs. In conclusion, the aggregation of porcine SCNT embryos at the four-cell stage could be a useful technique for improving the development rate and quality of porcine-cloned blastocysts and the derivation efficiency of porcine ntESCs.

Autophagy is a major intracellular pathway for the degradation and recycling of cytosolic components. Emerging evidence has demonstrated its crucial role during the embryo development of invertebrates and vertebrates. We recently demonstrated a massive activation of autophagy in Paracentrotus lividus embryos under cadmium stress conditions, and the existence of a temporal relationship between induced autophagy and apoptosis. Although there have been numerous studies on the role of autophagy in the development of different organisms, information on the autophagic process during oogenesis or at the start of development in marine invertebrates is very limited. Here we report our recent data on the occurrence of autophagy at these key phases of development. In order to investigate autophagy trends we performed in vivo assays to detect autophagolysomes, as well as in situ analysis with anti-LC3 antibody to detect autophagosomes before the fusion with lysosomes. From data generated through confocal laser scanning microscopy and quantification of autophagic signals we have drawn several unequivocal conclusions. The results showed a copious and rising number of autophagic organelles that had specific localization. Interestingly the increase in autophagy that occurred just after fertilization has been proved to be crucial for correct initiation of the developmental programme: irreversible developmental delays and morphologic anomalies were induced by short autophagic inhibition. This work focused on the sea urchin model system and corroborates evidence on the need for self-digestion during development, enriching the knowledge on autophagy, a biological mechanism belonging to evolutionarily different organisms.