Energy metabolism in pig oocytes and early embryos

Pig oocytes and embryos differ from those of other species in having a large quantity of endogenous lipid, a potential role for which has yet to be identified. In the present study, the hypothesis that endogenous triglyceride acts as a metabolic substrate during in vitro maturation and early embryo development was tested. Embryos were produced by in vitro fertilization (IVF) of in vitro-matured, abattoir-derived immature oocytes, cultured in medium NCSU23 up to the blastocyst stage. The triglyceride content of single oocytes and embryos was measured throughout development. Oxygen and glucose consumption and the formation of lactate were measured non-invasively over the same period, enabling total ATP production to be calculated. The triglyceride content of oocytes before maturation (135+/-4.9 ng) decreased by 13 ng (P<0.05) during in vitro maturation, but there was no apparent change in triglyceride content during embryo development (117.68 ng). Oxygen consumption was low throughout embryo cleavage before reaching a peak at the blastocyst stage (P<0.01), a pattern similar to that seen in other mammals studied. Glucose consumption and lactate production were also at a maximum at the blastocyst stage (P<0.05). These data indicate that pig oocytes may use endogenous triglyceride as an energy source during in vitro maturation and that most (91-97%) of the ATP produced during embryo development comes from oxidative phosphorylation. The high exogenous glucose concentration in NCSU23 (5.5 mmol l(-1)) may be needed to form pyruvate, which in turn, produces oxaloacetate, which is required to prime the tricarboxylic acid cycle. However, the reason for the high lipid content in early pig embryos remains to be elucidated.     

Replacement of nuclear protein by histone in pig sperm nuclei during in vitro fertilization

Sperm-specific nuclear protamines are dissociated before decondensation of sperm nuclei during fertilization in pigs. In the present study, replacement of nuclear protein by histone in boar spermatozoa during in vitro fertilization was evaluated by immunohistochemistry using anti-histone antibody. First, the specificity of the antibody used in this study was examined. Immunohistochemistry of the testes and epididymides indicated that somatic nuclei, but not elongated spermatids or maturing spermatozoa, were immunoreactive. Furthermore, immunoreaction was diminished after the antibody had been preincubated with unfractionated histone, indicating that the antibody was specific for the somatic nuclear histone. Immunohistochemistry of serial sections of oocytes, which were matured and co-cultured with boar spermatozoa for 2 to 6 h indicated that, at 2 to 3 h after insemination, penetrating sperm nuclei in the condensed state were not immunoreactive. At 4 to 5 h after insemination, some of the condensed sperm nuclei were immunoreactive in part or over the whole area of the nucleus, and all of the decondensing nuclei and male pronuclei were immunoreactive. At 6 h after insemination, the decondensing sperm nuclei and well-developed male pronuclei were immunoreactive. These results imply that, in pigs, remodelling of sperm nuclear protein from protamine to histone is initiated at the time of sperm penetration, before onset of decondensation and male pronuclear formation.     

Histone exchange activity and its correlation with histone acetylation status in porcine oocytes

In mammalian oocytes, histone H3 and histone H4 (H4) in the chromatin are highly acetylated at the germinal vesicle (GV) stage, and become globally deacetylated after GV breakdown (GVBD). Although nuclear core histones can be exchanged by cytoplasmic free histones in somatic cells, it remains unknown whether this is also the case in mammalian oocytes. In this study, we examined the histone exchange activity in maturing porcine oocytes before and after GVBD, and investigated the correlations between this activity and both the acetylation profile of the H4 N-terminal tail and the global histone acetylation level in the chromatin. We injected Flag-tagged H4 (H4-Flag) mRNA into GV oocytes, and found that the Flag signal was localized to the chromatin. We next injected mRNAs of mutated H4-Flag, which lack all acetylation sites and the whole N-terminal tail, and found that the H4 N-terminal tail and its modification were not necessary for histone incorporation into chromatin. Despite the lack of acetylation sites, the mutated H4-Flag mRNA injection did not decrease the acetylation level on the chromatin, indicating that the histone exchange occurs partially in the GV chromatin. In contrast to GV oocytes, the Flag signal was not detected on the chromatin after the injection of H4-Flag protein into the second meiotic metaphase oocytes. These results suggest that histone exchange activity changes during meiotic maturation in porcine oocytes, and that the acetylation profile of the H4 N-terminal tail has no effect on histone incorporation into chromatin and does not affect the global level of histone acetylation in it.     

Localization of CD9 in pig oocytes and its effects on sperm-egg interaction

CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm-oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm-oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 +/- 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 +/- 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm-oocyte interactions during fertilization.     

Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae

The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post‐translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone‐specific proteases. Here, we employed various biochemical methods to report histone H3‐specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3‐clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.     

Pairing and synapsis in oocytes from female fetuses with euploid and aneuploid chromosome complements

Only little is known about the meiotic prophase events in human oocytes, although some of them are involved in the origin of aneuploidies. Here, a broad study of the pairing and synaptic processes in 3263 human euploid and 2613 aneuploid oocytes (47,XX, +21 and 47,XX, +13), using different techniques and methods, is presented in order to elucidate the characteristics of this essential meiotic process. Our results reaffirm the existence of a common high efficiency in the pairing process leading to the obtainment of a bivalent for all chromosomes studied in euploid and aneuploid cases. Nevertheless, this high efficiency was insufficient to consistently produce trivalents in aneuploid oocytes. Trivalent 21 was only observed in 48.8% of the 47,XX, +21 pachytene-stage oocytes studied, and trivalent 13 was found in 68.7% of the 47,XX, +13 pachytene-stage oocytes analyzed. Our data confirm the hypothesis which suggests that in human oocytes the presence of an extra chromosome could interfere in bouquet dynamics. In addition, the pairing process of the X chromosome is altered in trisomic 21 oocytes, providing evidence of the influence that an extra chromosome 21 may cause meiotic progression.     

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes

Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified-warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.     

Role of ATRX in chromatin structure and function: implications for chromosome instability and human disease

Functional differentiation of chromatin structure is essential for the control of gene expression, nuclear architecture, and chromosome stability. Compelling evidence indicates that alterations in chromatin remodeling proteins play an important role in the pathogenesis of human disease. Among these, α-thalassemia mental retardation X-linked protein (ATRX) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres and telomeres as well as facultative heterochromatin on the murine inactive X chromosome. Mutations in human ATRX result in an X-linked neurodevelopmental condition with various degrees of gonadal dysgenesis (ATRX syndrome). Patients with ATRX syndrome may exhibit skewed X chromosome inactivation (XCI) patterns, and ATRX-deficient mice exhibit abnormal imprinted XCI in the trophoblast cell line. Non-random or skewed XCI can potentially affect both the onset and severity of X-linked disease. Notably, failure to establish epigenetic modifications associated with the inactive X chromosome (Xi) results in several conditions that exhibit genomic and chromosome instability such as fragile X syndrome as well as cancer development. Insight into the molecular mechanisms of ATRX function and its interacting partners in different tissues will no doubt contribute to our understanding of the pathogenesis of ATRX syndrome as well as the epigenetic origins of aneuploidy. In turn, this knowledge will be essential for the identification of novel drug targets and diagnostic tools for cancer progression as well as the therapeutic management of global epigenetic changes commonly associated with malignant neoplastic transformation.     

Independent activation of MAP kinase and MPF during the initiation of meiotic maturation in pig oocytes

Mitogen-activated protein (MAP) kinase is universally activated during oocyte maturation in all vertebrates studied to date. Its role in the resumption of meiosis and in the activation of maturation-promoting factor (MPF) remains unclear, especially in domestic species such as the pig. This study aimed to clarify the temporal and causal relationships between MAP kinase and MPF during meiotic maturation, particularly during the resumption of meiosis. Pig oocytes were matured synchronously in culture by treatment with cycloheximide. Kinase activities were analysed using a sensitive in vitro double-kinase assay and the specific MAP kinase pathway inhibitor U0126. MAP kinase and MPF were activated simultaneously at the time of germinal vesicle breakdown (GVBD; 6 h after removal of cycloheximide); they reached significant activity at 7 h (P < 0.05). The activities increased in parallel during GVBD (6-10 h) and peaked when the oocytes entered metaphase I (MI; 10 h). Whereas MAP kinase remained stable at peak activity thereafter, MPF activity significantly declined during the MI-MII transition (16-20 h) but increased to a second peak at MII (22 h). MAP kinase activity in denuded and cumulus-cell enclosed oocytes was completely inhibited by 20 and 80 mmicro mol U0126 l(-1), respectively. Oocytes without detectable MAP kinase activity underwent normal GVBD in terms of nuclear morphology and timing, although later meiotic stages were abnormal. The kinetics of MPF activity during GVBD were unaffected by U0126. This study has demonstrated that MAP kinase is activated simultaneously with MPF at GVBD, but that its activation is not essential for the activation of MPF nor for the resumption of the first meiosis in pig oocytes.     

Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.     

Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3

The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation. The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing. This has indicated that K. lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S. cerevisiae protein. The K. lactis TRP1 gene has been disrupted by substituting the S. cerevisiae URA3 gene for a large part of the TRP1 coding sequence. Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K. lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned. DNA sequencing has also shown that the K. lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively. Hybridization studies have shown that in common with S. cerevisiae, K. lactis has two copies of the histone H3 gene. Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.     

Physiological temperature variants and culture media modify meiotic progression and developmental potential of pig oocytes in vitro

Ovarian follicles in vivo are cooler than surrounding abdominal and ovarian tissues. This study investigated whether typical follicular temperatures influence the maturation and developmental potential of pig oocytes in vitro. Oocytes were synchronised at the germinal vesicle (GV) stage and incubated at 39, 37 or 35.5 degrees C. When compared with 39 degrees C, which is often used for in vitro studies, lower temperatures delayed spontaneous progression to the metaphase I and II (MI and MII) stages of meiosis. The MII was delayed by about 12 h per degrees C. All oocytes had normal morphology. Oocytes reaching GV breakdown (GVBD) at 39 degrees C were subsequently unaffected by cooling, demonstrating thermal sensitivity during the pre-GVBD stage only. Simultaneous assay of maturation-controlling kinases (maturation promoting factor (MPF) and MAPK) showed that cooling delayed kinase activation, provided it was applied prior to GVBD. Activity profiles remained coupled to the stage of meiosis. Neither enzyme was directly thermally sensitive over this temperature range. Following in vitro fertilisation, fewer blastocysts developed from embryos derived from 35.5 or 37 degrees C oocytes as compared with those from 39 degrees C oocytes. Manipulation of fertilisation timings to allow for delayed maturation showed that over-maturing or aging at lower temperatures compromises subsequent embryo development, despite normal nuclear maturation; the GV stage was again the thermally sensitive period. Cleavage rates were improved by the culture of oocytes with follicle-stimulating hormone (FSH) at 37 but not at 35.5 degrees C. Inclusion of 20% follicular fluid in the oocyte medium restored the blastocyst rate to that seen at higher temperatures. Thus, FSH and follicular fluid may allow oocytes to achieve normal developmental potential at in vivo temperatures.     

Localization of centromere proteins and their association with chromosomes and microtubules during meiotic maturation in pig oocytes

Centromere proteins (CENPs) are required for the attachment of microtubules to chromosomes. However, their structure and mechanism of action are not well understood, especially in mammalian meiosis. The present study was conducted to examine (i). whether a human nuclear centromere autoantibody can be used to localize the CENPs in pig oocytes and (ii). the dynamics of CENPs and their association with microtubules and chromosomes during meiosis in pigs. Oocytes at various stages were double-labelled for CENPs, chromosomes or microtubules and examined by confocal fluorescence microscopy. Quantification of tubulin and CENPs in the oocytes was determined by immunoblotting. CENPs were detected in all oocytes from germinal vesicle (GV) to metaphase II (MII) stages. The changes in the location were associated with chromosome movement and spindle formation. Tubulin was detected in the oocytes from GV to MII stages and no differences in content were observed. Two major CENPs at 80 kDa (CENP-B) and 50 kDa (CENP-D) were also found in the oocytes by the autoantibody and its content was significantly lower in the oocytes at GV stage compared with oocytes at other stages. These results indicate that the autoantibody used in this study can be used to detect CENPs in the kinetochores, and the proteins are expressed in pig oocytes at all stages during meiosis. As the localization of CENPs is associated with spindle formation and chromosome movement, CENPs may participate in cell cycle changes during meiosis in mammals.     

Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.     

Chromosome 18 pairing behavior in human trisomic oocytes. Presence of an extra chromosome extends bouquet stage

Little is known about the first meiotic prophase stages in the human female because these occur during fetal life, and only a few studies have addressed aneuploid human oocytes. In this paper, the synaptic process in the meiotic prophase in three 47, XX+18 cases is analyzed. A complete study of the dynamics of centromeres and telomeres, cohesin core and synapsis development in aneuploid female meiosis was performed. Investigation of chromosome dynamics in prophase of trisomy 18 oocytes show that these events follow the major patterns seen earlier in euploid oocytes. However, there is a significant delay in the resolution of bouquet topology which could relate to the presence of a surplus chromosome 18 axial element in zygotene oocytes. Pachytene oocytes displayed normal synapsis among the three chromosome 18s. However, in some oocytes the surplus chromosome 18 core was aligned to the bivalent 18. As ataxia telangiectasia and Rad3 related kinase (ATR) has been described as a marker for late-pairing chromosomes in mice, ATR distribution was analyzed in human meiocytes--spermatocytes, euploid oocytes and trisomic oocytes. In contrast to the observations made in mice, no preferential staining for late-pairing chromosomes was observed in humans. In the cases studied, bivalent synapses progressed as in a normal ovary, contrasting with the hypothesis that a surplus chromosome can modify pairing of other chromosomes.     

Lysis of oocytes by bovine sperm and seminal plasma

This study examined the lytic activity of bovine seminal plasma on zona-free oocytes commonly used to assess bovine capacitation. Exposure of hamster oocytes (intact or zona-free) to undiluted seminal plasma resulted in lysis within 5 min. With intact oocytes, 1:5 to 1:20 seminal plasma resulted in swelling of the oocytes. With zona-free oocytes, seminal plasma (1:0 to 1:1000) resulted in lysis within 1 min to 3 h depending on dilution. Heating seminal plasma to inactivate complement did not reduce lytic activity, but boiling destroyed it. Lytic activity was present in seminal plasma from vasectomized bulls and in seminal vesicle fluid. After elution of seminal plasma through Sephadex G-200, lytic activity was only associated with the fraction containing proteins of 200,000 to 45,000 dal. Lytic activity remained with washed capacitated bull sperm only when 10(7) sperm/ml or more were coincubated with zona-free oocytes at 37 degrees C for 3 h. In conclusion, both bovine seminal plasma and capacitated sperm were lytic in the zona-free hamster oocyte assay. The results may explain why so few sperm are normally found at the site of fertilization.