For decades, scientists have considered that female mammals are born with a lifetime reserve of oocytes in the ovary, irrevocably fated to decline after birth. However, controversy in the matter of the possible presence of oocytes and granulosa cells that originate from stem cells in the adult mammalian ovaries has been expanded. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of neo-oogenesis, who claim that germline stem cells (GSCs) exist in the ovarian surface epithelium (OSE) or the bone marrow (BM). Differentiation of ovarian stem cells (OSCs) into oocytes, fibroblast-like cells, granulosa phenotype, neural and mesenchymal type cells and generation of germ cells from OSCs under the contribution of an OSC niche that consists of immune system-related cells and hormonal signalling has been claimed. Although these arguments have met with intense suspicion, their confirmation would necessitate the revision of the current classic knowledge of female reproductive biology.

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

Round spermatid injection (ROSI) into mammalian oocytes can result in the development of viable embryos and offspring. One current limitation to this technique is the identification of suitable round spermatids. In the current paper, round spermatids were selected from testicular cells with phase contrast microscopy (PCM) and fluorescence-activated cell sorting (FACS), and ROSI was performed in two strains of mice. The rates of fertilization, embryonic development and offspring achieved were the same in all strains. Significantly, round spermatids selected by PCM and FACS were effectively used to rescue the infertile Pten-null mouse. The current results indicate that FACS selection of round spermatids can not only provide high-purity and viable round spermatids for use in ROSI, but also has no harmful effects on the developmental capacity of subsequently fertilized embryos. It was concluded that round spermatids selected by FACS are useful for mouse strain rederivation and rescue of infertile males; ROSI should be considered as a powerful addition to the armamentarium of assisted reproduction techniques applicable in the mouse.

Initiation of sperm motility in urodeles, which is induced by a sperm motility-initiating substance (SMIS) in the sequestered granules on the surface of egg jelly, is mediated by the acrosome reaction (AR), which is triggered by an AR-inducing substance (ARIS) on a sheet-like structure. Details of the unique process of the interaction between egg jelly and sperm in these species is still unclear. The current study showed the fine structure of egg jelly in the newt Cynops pyrrhogaster, a urodele species, revealing that its outer surface was covered by a sheet-like structure of approximately 0.29 μm in thickness. Granules of approximately 2 μm in diameter with small particles of approximately 54 nm were attached to its surface and distributed inhomogeneously just beneath the sheet-like structure. Emission spectrometry revealed that the Ca2+ concentration was maintained at a high level compared with that of the blood plasma and the vas deferens fluid, suggesting that egg jelly is a reliable source of Ca2+ for the sperm–egg interaction. Blockers of the T-type voltage-dependent Ca2+ channel (VDCC), but not the L-type VDCC, inhibited both AR and initiation of sperm motility. Conversely, Ni+, which affects the α1 H subunit of T-type VDCC, only inhibited the initiation of sperm motility. These data suggest that, in response to ARIS and SMIS, sequential gating of distinct Ca2+ channels occurs in the AR, followed by the initiation of sperm motility on the surface of the egg jelly in C. pyrrhogaster at fertilization.

The aim of the present study was to examine the effects of brilliant cresyl blue (BCB) staining on mitochondrial functions in porcine oocytes. Cumulus–oocyte complexes (COCs) collected from slaughterhouse-derived porcine ovaries were cultured with (13 μM) or without (0 μM, control) BCB for 60 min. Mitochondrial functions in oocytes were examined immediately after staining or after in vitro maturation. The BCB-stained oocytes produced reactive oxygen species (ROS) at higher levels than control oocytes immediately after staining (2.2-fold, P < 0.001) and after maturation (1.7-fold, P < 0.001). The adenosine triphosphate (ATP) content and mitochondrial membrane potential (MMP) in oocytes were similar for the two groups immediately after staining. However, ATP and relative MMP levels were significantly (P < 0.05) lower in BCB-treated oocytes than in the control (2.18 versus 2.83 pM and 0.82 versus 1.0, respectively). There was no difference in mitochondrial DNA copy number between the two groups after maturation. The ATP content in early developmental stage embryos (3 days after parthenogenetic activation) was lower in the BCB-stained group than that in the control group but the difference was not significant. In conclusion, BCB staining of oocytes at the immature stage compromises mitochondrial functions throughout oocyte maturation, but function is restored during early embryo development.

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 μm in thickness with eight pore canals/μm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P<0.05). No statistical differences were found for blastocyst cell numbers. The mRNA expression for the EGR1 gene was down-regulated eight-fold in blastocysts that had been produced in vitro compared with their in vivo counterparts. Gene expression profiles for IGF2R, SLC2A8, COX2, DNMT3B and PCK2 did not differ among experimental groups. Bovine testis satellite I and Bos taurus alpha satellite methylation profiles from cAMP30aspiration protocol-derived blastocysts were similar to patterns that were observed in their in vivo equivalents (P > 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

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 expression of nine serotonin (5-HT) receptor transcripts was studied using reverse transcription polymerase chain reaction (RT-PCR) in germ cells, cleavage and gastrulation stages of Japanese quail, and qPCR for 5-HT3 and 5-HT4 receptors in oocytes and embryos. We show the presence/absence of nine serotonin transcripts known in birds for receptors 5-HT1A, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A in avian germ cells and early embryos. The absence of 5-HT3 and 5-HT5A in primordial germ cells and of 5-HT3 and 5-HT7A in sperm is characteristic. All transcripts appeared in oocytes at all stages (except for 5-HT3 and 5-HT5A transcripts) and all were present in cleaving embryos and at gastrulation, except for 5-HT3, which was permanently observed as late as in stage 4. Interestingly, 5-HT3 and 5-HT5A receptors accumulated in 3-mm and F1 oocytes but were degraded at ovulation and started to be re-transcribed in cleavage stage II embryos and beyond. The selective appearance of 5-HT receptors in germ cells and early embryos supports the hypothesis that serotonin may act as a signalling molecule at early stages of germ line and embryo differentiation via individual receptors present during different stages, when specialized communication systems are not yet developed.

The sesquiterpene lactones (STLs) are a large class of plant secondary metabolites that are generally found in the Asteraceae family and that have high diversity with respect to chemical structure as well as biological activity. STLs have been classified into different groups, such as guaianolides, germacranolides, and melampolides etc., based on their carboxylic skeleton. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under the stimulus of progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. The purpose of this work was to determine whether sesquiterpene lactones from the germacranolide and melampolide groups act as inhibitor agents on the meiosis of amphibian oocytes in vitro. Results for germacranolides indicated that the addition of deoxyelephantopins caused a high degree of inhibition and that minimolide showed a moderate inhibitory effect, whereas glaucolide A was inactive. Furthermore, the addition of melampolides (uvedalin, enhydrin, polymatin A and polymatin B) showed inhibitory effects. For enhydrin and uvedalin, inhibitory effects were observed at the higher concentrations assayed. The results of this study suggest that the inhibitory activity of the tested sesquiterpene lactones on the meiosis of Rhinella arenarum oocytes is not dependent on the group to which they belong, i.e. not on the carboxylic skeleton, but probably due to the arrangement and type of function groups present in the molecules. All assayed lactones in the germacranolide group showed low toxicity. In contrast, important differences in toxicity were observed for lactones from the melampolide group: enhydrin and uvedalin showed low toxicity, but polymatin A and B were highly toxic.

The aim of the present study was to clarify the efficacy of the well of the well (WOW) culture system for a small number of embryos and the effect of number of adjacent embryos in a WOW dish on blastocyst development. In conventional droplet culture, embryos in the small-number group (5–6 embryos/droplet) showed low blastocyst development compared with a control group (25–26 embryos/droplet). However, small and large numbers of embryos (5–6 and 25 embryos, respectively) in a WOW dish showed no significant differences in cleavage, blastocyst rates, and mean cell number in blastocysts compared with the control group (25–30 embryos/droplet). In addition, the number of adjacent embryos in a WOW dish did not affect the development to blastocysts and cell number in blastocysts. In conclusion, a WOW dish can provide high and stable blastocyst development in small group culture wherever embryos are placed in microwells of the WOW dish.

Oocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal–vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal–vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

Emerging organic contaminants have been monitored in stream waters, raw and finished waters and wastewater effluents. Most of these contaminants, such as epigenetic substances, have been detected at very low levels. Unfortunately, their complete monitoring and/or removal are very difficult, given the increasing presence of new contaminants and due to analytical and economic considerations. For this reason, bioindicators are used as an alternative to monitor their presence. To this end, zebrafish is being used to assess certain contaminants in water quality studies.

As our long-term aim is to determine if zebrafish (Danio rerio) can be used to detect environmental epigenetic factors in drinking waters with effects on human reproduction, an initial question is whether the chorion could interfere with the possible action of epigenetic factors in two reproductive events: genital ridge formation and migration of the primordial germ cells (PGCs) to these genital ridges.

In the first experiment, we attempted to partially degrade the chorion of mid blastula transition (MBT) embryos with pronase, with acceptable survival rates at 5 days post fertilisation (dpf), with the group exposed for 15 min giving the best survival results. As denuded early embryos require a specific culture medium, in the next experiment embryo survival was evaluated when they were cultured up to 5 dpf in drinking waters from six different sources. Results showed a negative effect on embryo survival at 5 dpf from several waters but not in others, thus distorting the survival outcomes. These results suggest using embryos with the chorion intact from the outset when drinking waters from different sources are to be tested.

Cryopreservation has not been used successfully to preserve fish embryos, although chilling techniques have been used with good results. The aim of this study was to chill Piaractus brachypomus embryos at – 10°C for various storage times. Embryos at the following ontogenetic stages were used: blastoderm – 1.2 hours post-fertilization (hpf); epiboly – 5 hpf; blastopore closure – 8 hpf; and appearance of the optic vesicle – 13 hpf. One hundred embryos were selected from each ontogenetic stage and chilled at – 10°C for 6 or 10 h. The results were analysed using analysis of variance (ANOVA) and Tukey's test at a 5% significance level. A significantly greater number of completely developed live larvae were observed following embryonic treatment with a cryoprotectant solution that contained 17.5% sucrose and 10% methanol. There was no survival for embryos cooled at – 10°C in initial developmental stages (1, 2 and 5 h hpf). Furthermore, higher survival rates were observed when embryos were treated at more advanced developmental stages (8 and 13 hpf). Therefore, P. brachypomus embryos at the blastopore-closure (8 hpf) or appearance-of-optic-vesicle (13 hpf) stages should be used for embryo chilling protocols and chilling should be performed using a 17.5% sucrose with a 10% methanol solution at – 10°C for up to 6 h. The best results were obtained with 13-hpf and 8-hpf embryos and cooling at 6 h of storage.

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.

Primary mesenchyme cells (PMCs) are skeletogenenic cells that produce a calcareous endoskeleton in developing sea urchin larvae. The PMCs fuse to form a cavity in which spicule matrix proteins and calcium are secreted forming the mineralized spicule. In this study, living sea urchin embryos were stained with fluorescently conjugated wheat germ agglutinin, a lectin that preferentially binds to PMCs, and the redistribution of this fluorescent tag was examined during sea urchin development. Initially, fluorescence was associated primarily with the surface of PMCs. Subsequently, the fluorescent label redistributed to intracellular vesicles in the PMCs. As the larval skeleton developed, intracellular granular staining diminished and fluorescence appeared in the spicules. Spicules that were cleaned to remove membranous material associated with the surface exhibited bright fluorescence, which indicated that fluorescently labelled lectin had been incorporated into the spicule matrix. The results provide evidence for a cellular pathway in which material is taken up at the cell surface, sequestered in intracellular vesicles and then incorporated into the developing spicule.