This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150–200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal–Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of β-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.

A histological characterization of gonadal development in the tetra Astyanax bimaculatus was performed, aimed at determining its reproductive cycle in streams localized inside the Amazonian forest. Collections were carried out monthly from August 2017 to July 2018 at the Zoobotânica Foundation of Marabá, PA. Collected specimens were weighed and measured, and their gonads and liver were removed and weighed to calculate gonadosomatic and hepatosomatic indexes. Gonads were fixed and treated for routine histology for light microscopy. Materials were stained with toluidine blue and haematoxylin and eosin. The Amazonian A. bimaculatus species presented two reproductive periods in the year, one at the end of the winter season and another during the summer. Females showed an asynchronous development of their oocytes and only two reproductive phases of development were observed during the whole period ‘developing’ and ‘spawning capable’. Males presented cystic spermatogenesis, with an anastomosing tubular testis containing spermatogonia spread along the germinal epithelium (unrestricted spermatogonial). These morphological characteristics are considered phylogenetically more primitive. Male specimens were observed to have five different phases during the period: immature, initial maturation, mid maturation, final maturation and regression. The huge fluctuations in Amazonian streams was observed, in which water volumes varied considerably across seasons, culminating even in total drought. In spite of this, A. bimaculatus could be found during all seasons, showing its impressive reproductive adaptation to its conditions.

In this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

The low efficiency of somatic cell nuclear transfer may be related to the ultrastructural deviations of reconstructed embryos. The present study investigated ultrastructural differences between in vivo-produced and cloned goat embryos, including intra- and interspecies embryos. Goat ear fibroblast cells were used as donors, while the enucleated bovine and goat oocytes matured in vitro as recipients. Goat–goat (GG), goat–cattle (GC) and goat in vivo-produced embryos at the 2-cell, 4-cell, 8-cell and 16-cell stages were compared using transmission electron microscopy. These results showed that the three types of embryos had a similar tendency for mitochondrial change. Nevertheless, changes in GG embryos were more similar to changes in in vivo-produced embryos than were GC embryos, which had more extreme mitochondrial deviation. The results indicate the effects of the cytoplast on mitochondria development. The zona pellucida (ZP) in all three types of embryos became thinner and ZP pores in both GC and GG embryos showed an increased rate of development, especially for GC embryos, while in vivo-produced embryos had smooth ZP. The Golgi apparatus (Gi) and rough endoplasmic reticulum (RER) of the two reconstructed embryos became apparent at the 8-cell stage, as was found for in vivo embryos. The results showed that the excretion of reconstructed embryos was activated on time. Lipid droplets (LD) of GC and GG embryos became bigger, and congregated. In in vivo-produced embryos LD changed little in volume and dispersed gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere illuminated that the nucleus function of reconstructed embryos was partly changed. In addition, at a later stage in GC embryos the nuclear envelope displayed infoldings and the chromatin was concentrated, implying that the blastomeres had an obvious trend towards apoptosis. The gap junctions of the three types of embryos changed differently and GG and GC embryos had bigger perivitelline and intercellular spaces than did in vivo-produced embryos. These results are indicative of normal intercellular communication at an early stage, but this became weaker in later stages in reconstructed embryos. In conclusion, inter- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe destruction. These ultrastructural deviations might contribute to the compromised developmental potential of reconstructed embryos.

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.

Transgenes integrated into mammalian cells are silenced rapidly. This phenomenon correlates with repressed chromatin structure marked by histone hypoacetylation. This study investigated the effect of trichostatin A (TSA; a histone-deacetylase inhibitor) on EGFP expression in transfected cells and embryonic development after somatic cell nuclear transfer (SCNT). Porcine adult fibroblasts were transfected with a pEGFP-C1 vector. Then transfected cells, donor cells for SCNT, were pretreated with TSA, with the untreated cells being used as the control. Expression of EGFP in donor cells and reconstructed embryos was detected when exposed to blue light. Results showed that the percentage of EGFP-positive cells significantly increased when the transfected cells were treated with TSA and the increased expression of EGFP was sustained to at least the morula stage. In addition, the cytotoxic effect of TSA on the transfected cells was dose dependent. In conclusion, TSA can rescue the silenced EGFP gene. Even after transferring the TSA-treated cells to enucleated recipient oocytes, TSA retained the ability to rescue a silenced EGFP gene. In addition, TSA had an impact on cell proliferation.

Methomyl is a widely used carbamate insecticide and environmental oestrogen that has adverse effects on the reproductive system. However, there have been no reports on the effect of methomyl on early embryos in mammals. In this study, we explored the effect of methomyl exposure on the quality of early embryonic development in mice and the possible mechanisms. During in vitro culture, different concentrations of methomyl (10, 20, 30 and 35 μM) were added to mouse zygote medium. The results showed that methomyl had an adverse effect on early embryonic development. Compared with the control group, the addition of 30 μM methomyl significantly reduced the rate of early embryo blastocyst formation. Methomyl exposure can increase oxidative stress and impair mitochondrial function, which may be the cause of blastocyst formation. In addition, we found that methomyl exposure promoted apoptosis and autophagy in mouse blastocysts. The toxic effect of methomyl on early embryos may be the result of oxidative stress induction. Taken together, our results indicate that methomyl can cause embryonic development defects in mice, thereby reducing the quality of early embryo development.

The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6–8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.

Intracellular membranes isolated from fully grown immature oocytes, mature oocytes (eggs) and activated eggs of Xenopus laevis were fractionated through a discontinuous sucrose density gradient into light, intermediate and heavy fractions. Electron microscopy showed that the light and intermediate fractions consisted mainly of smooth membranes, while the heavy fraction consisted mainly of rough membranes and mitochondria. Variations in the proteins associate with samples taken at different stages were observed by SDS-PAGE. The following differences were consistently observed: a 200 kDa protein was present only in the intermediate fraction of activated eggs, 29 and 44 kDa proteins were present only in the intermediate fractions of immature oocytes and activated eggs, and 120 and 145 kDa proteins were present only in the heavy fractions of mature oocytes and activated eggs. Examination of Western blots showed that cyclins A and B2 did not associate with membrane fractions at any stage of meiosis. Instead, cyclin A was present in the cytosols of mature oocytes and cyclin B2 was present in the cytosols of immature and mature oocytes. c-mos protein was detected in the cytosols and occasionally in the light fractions of mature oocytes and activated eggs. While α- and β-tubulins were detected in the light and intermediate fractions at all the stages of meiosis examined, only β-tubulin was present in the heavy fraction. βtubulin present in the heavy fraction was detected only at interphase, i.e. in immature oocutes and activated eggs, and not in mature oocytes. Using immunogold labelling we confirmed these results and found evidence to suggest that β-tubulin associates with the rough endoplasmic reticulum of interphase cells by a linking protein.

DNA methylation is one of the proposed biochemical mechanisms involved in cell differentiation and in genomic imprinting, and DNA methyltransferase (DMT) is a key enzyme in the embryo since mutation of its gene is lethal early in development. In order to verify that non-viability of uniparental embryos was not due to a defect in the regulation of DMT activity, we compared the metabolism of methylation in parthenogenetic embryos (maternal genome) and in fertilised embryos (maternal and paternal genomes). As regards total methylation, estimated by a measure of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) formation, no significant difference was found between the two kinds of embryos during preimplantation development. Mean values were 4.5 ± 0.6 fmol (SAM +SAH)/h per 2-cell embryo and 0.40 ± 0.05 fmol SAH/h per 2-cell embryo, i.e. a SAH/(SAM + SAH) ratio of 9%; there was no detectable SAH formation in blastocysts. The same observation can be made for DMT activity, with mean values of: 7.8 fmol/h per oocyte, 8.5 fmol/h per 2-cell embryo, 6.1 fmol/h per 4-cell embryo, 4.1 fmol/h per morula, and no detectable activity in blastocysts. Total methylation as well as DNA methylation is characterised by a progressive drop in activity during preimplantation development.

RNA transcription, processing and translation are fundamental molecular processes required for development, growth and cell viability. Towards the functional annotation of the genome, we are engaged in a reverse genetic screen using mammalian preimplantation embryos as a model system. Here we report the essential function of four RNA processing/splicing factors (Sf3b14, Sf3b1, Rpl7l1, and Rrp7a) and show that each of these genes is required for blastocyst formation in the mouse. As very little information is known about these genes, we characterized their normal expression and localization in mouse embryos as well as phenotypic analysis of loss of function during preimplantation development. Functional knockdown of each gene product results in normal morula development but there is failure to form a blastocoel cavity or morphologically differentiated trophectoderm. We show that zygotic genome activation does occur as well as initial lineage specification in the absence of each factor. Consistent with a role in RNA splicing, we demonstrate that the absence of Sf3b14 and Sf3b1 in 8-cell and morula-stage embryos results in a specific reduction of intron containing transcripts, but no reduction single-exon genes. Taken together, we show critical developmental and molecular requirements of Sf3b14, Sf3b1, Rpl7l1, and Rrp7a during mammalian preimplantation.

The aim of this study was to describe the embryonic and larval development of discus fish (Symphysodon aequifasciatus), and to determine the time required, hours post fertilization (hpf), for the emergence of various structures. To describe embryonic and larval stages, observations were made at regular periods under an optical microscope and images were taken with a digital camera attached to a microscope. The average temperature reached in the experimental tank was 27.9°C. Important facts in embryonic and larval development are described, such as the closure of the blastopore, which occurred at 31.5 hpf; a period of eruption, which occurred at 58.5 hpf; and a mouth opening, which occurred at 90.5 hpf; and larvae that exhibited oriented swimming and eating exogenous food at 136.5 hpf.

Environmental hypoxia exposure causes fertility problems in human and animals. Compelling evidence suggests that chronic hypoxia impairs spermatogenesis and reduces sperm motility. However, it is unclear whether paternal hypoxic exposure affects fertilization and early embryo development. In the present study, we exposed male mice to high altitude (3200 m above sea level) for 7 or 60 days to evaluate the effects of hypoxia on sperm quality, zygotic DNA methylation and blastocyst formation. Compared with age-matched controls, hypoxia-treated males exhibited reduced fertility after mating with normoxic females as a result of defects in sperm motility and function. Results of in vitro fertilization (IVF) experiments revealed that 60 days’ exposure significantly reduced cleavage and blastocyst rates by 30% and 70%, respectively. Immunohistochemical staining of pronuclear formation indicated that the pronuclear formation process was disturbed and expression of imprinted genes was reduced in early embryos after paternal hypoxia. Overall, the findings of this study suggested that exposing male mice to hypoxia impaired sperm function and affected key events during early embryo development in mammals.

Embryos that are produced in vitro frequently present epigenetic modifications. However, maternal supplementation with folic acid (FA) may improve oocyte maturation and embryo development, preventing epigenetic errors in the offspring. We sought to evaluate the influence of FA supplementation during in vitro maturation of grade I (GI) and grade III (GIII) bovine oocytes on embryo production rate and the expression of IGF2 and KCNQ1OT1 genes. The oocytes were matured in vitro with different concentrations of FA (0, 10, 30 and 100 μM), followed by in vitro fertilization and embryo culture. On the seventh day (D7) of culture, embryo production was evaluated and gene expression was measured using real-time qPCR. Supplementation with 10 μM of FA did not affect embryo production for GI and GIII oocytes. Moderate supplementation (30 μM) seemed to be a positive influence, increasing embryo production for GIII (P = 0.012), while the highest dose (100 μM) reduced embryo production (P = 0.010) for GI, and IGF2 expression was not detected. In GIII, only embryos whose oocyte maturation was not supplemented with FA demonstrated detected IGF2 expression. The lowest concentration of FA (10 μM) reduced KCNQ1OT1 expression (P = 0.05) on embryos from GIII oocytes. Different FA concentrations induced different effects on bovine embryo production and gene expression that was related to oocyte quality. Despite the epigenetic effects of FA, supplementation seems to be a promising factor to improve bovine embryo production if used carefully, as concentration is an important factor, especially in oocytes with impaired quality.

CD9 is a member of the tetraspanin superfamily proteins and is the only protein on the mouse oocyte which is known to be indispensable in sperm–egg fusion. Here, using indirect immunofluorescence we show that CD9 appears on the oolemma during the early stages of the growth of the oocyte, when it measures 13–22 μm in diameter. When the oocyte reaches a diameter of 17–22 μm, the density of CD9 in its oolemma is similar to the density of this protein in the cell membrane of the fully grown secondary oocyte. The appearance of CD9 in growing oocytes correlates with the previously reported time of the acquisition of fusibility between the spermatozoon and the egg. Accordingly we propose that during oogenesis the development of the ability of the oolemma to fuse with sperm may be regulated by synthesis of CD9 by the oocyte.

The exposure of oocytes to heat stress during the maturation process results in harmful effects to their internal organelles, low fertilization capability and higher embryonic losses. In the present experiment the effect of heat shock (HS) during the maturation process was assessed. In Assay 1, oocytes from winter (December–March; n = 100) and summer (June–September; n = 100) months were collected and matured to analyse their HS tolerance. Total RNA was extracted from matured oocytes and cDNA synthesis was performed, followed by qPCR for selected genes (Cx43, CDH1, DNMT1, HSPA14), compared with two reference genes (GAPDH and SDHA). In Assay 2, oocytes collected during the winter were subjected to kinetic HS by stressing them at 39.5°C for 6, 12, 18 or 24 h and were afterwards matured at control temperature (38.5°C), and subsequently subjected to the previously described gene analysis procedure. Results of Assay 1 show that summer-collected oocytes exhibited lower maturation rate than winter-collected oocytes, which may be due to the down-regulation of the HSPA 14 gene. Assay 2 showed that 6 h of HS had no effect on gene regulation. CDH1 and DNMT1 up-regulation was observed starting at 12 h, which may represent the effect of heat shock on oocyte development.

We evaluated the effect of glycerol on the perinuclear theca (PT) of boar sperm. Samples from six ejaculates obtained from three different boars were incubated in the detergent Brij 36-T. Spermatozoa were treated with a glycerol concentration of either 2 or 4%, and incubated for 10 or 30 min; two other samples were treated with protease inhibitors (PI; leupeptin or an inhibitor commercial cocktail), mixed with 4% glycerol, and incubated for 30 min. A third glycerol-free group was used as the control. The samples were processed for electron microscopy evaluation. The PT remained intact in 78% of the control samples while, after addition of glycerol for 30 min, the proportion of spermatozoa with disrupted or absent PT increased (P < 0.05). PT was preserved in PI samples, but PT changes increased (P < 0.05). Differences due to treatment with glycerol (2 or 4%) at 10 or 30 min were not observed. These results show, to our knowledge for the first time, the adverse effect of glycerol on the integrity of the PT.

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.