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The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 μM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 μs) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5–81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2–32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.
Recent interest in the initial phases of ovarian follicular formation and development has lead to a number of publications in this area, most of which address the autocrine and paracrine factors involved in primordial follicle activation to primary follicle. Primordial follicle assembly (first step in follicle formation) determines the lifetime supply of primordial follicles and remains a poorly understood phenomenon. Despite a number of recent articles that are concentrating on immuno-histochemistry, basic steps in the process are not clear. Hence, we feel it is time to take a step back and see what is available in the literature and identify the gaps in which future research about primordial follicle assembly in humans needs to be directed.
Previous studies showed that the addition of a growth factor to the culture medium could modulate embryo development. The possible secretion of different factors to the culture medium by the embryo itself, however, has been poorly evaluated. The present study was designed to investigate: (1) the influence of single or group culture on the development of 2-cell mouse embryos (strain CD-1) to the blastocyst stage; (2) the release of granulocyte–macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) into the culture medium by the embryo; and (3) the levels of GM-CSF and SCF in the culture medium from both single and group embryos. Two-cell CD-1 mouse embryos were cultured for 96 h singly or in groups of five embryos per drop. GM-CSF and SCF were assayed by ELISA in the complete culture medium. It was found that embryos cultured in groups gave a higher percentage of total blastocyst formation and hatched blastocyst when compared with single embryo culture. The mouse embryos secreted GM-CSF and SCF to the culture medium. The concentration of these cytokines is significantly higher in the group cultures than the level found in single cultures. In conclusion, mouse embryos in culture secrete GM-CSF and SCF to the culture medium and the concentration of these cytokines increases during communal culture. These factors may be operating in both autocrine and paracrine pathways to modulate embryo development during in vitro culture.
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.
During oocyte maturation, the cumulus–oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.
Oogenesis is a critical event in the formation of female gametes, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the chromatin architecture in newt oocytes. Using fluorescence microscopy, as well as transmission electron microscopy (TEM), immunohistochemical method and RE-ChIP assay, some peculiar aspects of chromatin and chromosome organization and evolution in crested newt oogenesis were investigated. We focussed our investigations on detection of certain epigenetic modifications (H4 hyperacetylation, H2A ubiquitinylation and cytosine methylation) at the rRNA gene (18S–5.8S–28S) promoter region. Our findings suggest that there is an involvement of some epigenetic modifications as well as of linker histone variants in chromatin architecture dynamics during crested newt oogenesis.
The amelogenin (AMEL) gene exists on both sex chromosomes of various mammalian species and the length and sequence of the noncoding regions differ between the two chromosome-specific alleles. Because both forms can be amplified using a single primer set, the use of AMEL in polymerase chain reaction (PCR)-based methods has facilitated sex identification in various mammalian species, including cattle, sheep and humans. In this study, we designed PCR primers to yield different-sized products from the AMEL genes on the X (AMELX) and Y (AMELY) chromosomes of pigs. PCR amplification of genomic DNA samples collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc and Berkshire; Chinese breeds: Meishan and Jinhua and their crossbreeds) yielded the expected products. For all breeds, DNA from male pigs produced two bands (520 and 350 bp; AMELX and AMELY, respectively), whereas samples from female pigs generated only the 520 bp product. We then tested the use of PCR of AMEL for sex identification of in vitro-produced (IVP) porcine embryos sampled at 2 or 5 to 6 days after fertilization; germinal vesicle (GV)-stage oocytes and electroactivated embryos were used as controls. More than 88% of the GV-stage oocytes and electroactivated embryos yielded a single 520 bp single band and about 50% of the IVP embryos tested produced both bands. Our findings show that PCR analysis of the AMEL gene is reliable for sex identification of pigs and porcine embryos.
Pseudoplatystoma coruscans is a very popular species for tropical fish culture as it has boneless meat of delicate taste and firm texture. Few studies on fish reproductive biology refer to the morphological features of eggs. The goal, therefore, of this present work was to perform a structural and ultrastructural analysis of fertilization and embryonic development in P. coruscans. The incubation period, from fertilization to hatching, lasts 13 h at 28/29 °C and 18 h at 27 °C. The oocytes had a mean diameter of 0.95 mm and hatched larvae were 2.55 mm in diameter. Analysing their development, we observed round, yellow oocytes that bore a double chorion membrane and a single micropyle. At 10 s after fertilization, several spermatozoa were detected attached to the oocyte surface. After 1 min of development, a fertilization cone that obstructed the micropyle could be observed. Segmentation started between 20 and 30 min after fertilization, when the egg cell was then formed. The first cleavage occurred between 30 and 45 min after fertilization, prior to reaching the morula stage (75 and 90 min after fertilization). The epiboly movement started at 120 and 180 min after fertilization and ended at 360 and 480 min after fertilization. Differentiation between cephalic and caudal region was detected after 420 and 600 min after fertilization and larvae hatched between 780 and 1080 min after fertilization. Seven main embryonic development stages were identified: egg cell, cleavage, morula, blastula, gastrula, segmentation with differentiation between cephalic and caudal regions, and hatching.
Recent revelations in the human reproductive process have fuelled much interest in this field of study. In particular, the once prevailing view of large numbers of ejaculated sperms racing towards the egg has been refuted recently. This is opposed to the current views derived from numerous clinical findings that state that only a very small number of sperms will ever enter the oviduct. It is believed that these few sperms must have been guided to make the long, tedious and obstructed journey to the egg. For a mature spermatozoon, its hyperactivated swimming motility upon capacitation plays an important role in the fertilization of a mature egg. Likewise, the female genital tract also provides guiding mechanisms to complement the survival of normal hydrodynamic profile sperms and thus promotes an eventual sperm–egg interaction. Understanding these mechanisms can be essential for the derivation of assisted conception techniques especially those in vitro. With the aid of computational models and simulation, suitability and effectiveness of novel assisted conception methodology can be assessed, particularly for those yet to be ready for clinical trials. This review discusses the possible bioengineering models and the mechanisms by which human spermatozoa are guided to the egg.
Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron–ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron–ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.