Energy metabolism disorders during in vitro maturation of bovine cumulus-oocyte complexes interfere with blastocyst quality and metabolism.

Glucose and fatty acids (FA) metabolism disturbances during oocyte in vitro maturation (IVM) affect their metabolism and surrounding cumulus cells, but only inhibition of glucose metabolism decreases embryo culture efficiency. Therefore, the present experiment aimed to reveal if glucose or FA metabolism inhibition leads to the disruption of embryo developmental potential, and to characterize the metabolic landscape of embryos reaching the blastocyst stage. Inhibitors of glucose (IO + DHEA) or FA (ETOMOXIR) metabolism were applied during IVM, and the control group was matured under standard conditions. Blastocysts obtained from experimental and control groups were analyzed with regard to lipidome and metabolome (mass spectrometry), transcriptome (RNA-Seq) and fluorescence lipid droplets staining (BODIPY). We showed that inhibition of glucose and fatty acid metabolism leads to cellular stress response compromising the quality of preimplantation embryos. The inhibition of energy metabolism affects membrane fluidity as well as downregulates fatty acids biosynthesis and gene expression of trophectoderm cell line markers. Therefore, we conclude that oocyte maturation environment exerts a substantial effect on preimplantation development programming at cellular and molecular levels.

Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer's Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer's Disease.

BACKGROUND: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer's disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. OBJECTIVE: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-ß (Aß) accumulation, and cognitive/neuromotor performance in mice. METHODS: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AßPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aß was quantified by western blotting (brains) and by confocal microscopy (cells). RESULTS: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AßPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aß was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aß without affecting AßPP. CONCLUSIONS: Our findings show that Blmh interacts with AßPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aß accumulation and cognitive/neuromotor deficits.

Species and embryo genome origin affect lipid droplets in preimplantation embryos.

Mammalian embryo development is affected by multiple metabolism processes, among which energy metabolism seems to be crucial. Therefore the ability and the scale of lipids storage in different preimplantation stages might affect embryos quality. The aim of the present studies was to show a complex characterization of lipid droplets (LD) during subsequent embryo developmental stages. It was performed on two species (bovine and porcine) as well as on embryos with different embryo origin [after in vitro fertilization (IVF) and after parthenogenetic activation (PA)]. Embryos after IVF/PA were collected at precise time points of development at the following stages: zygote, 2-cell, 4-cell, 8/16-cell, morula, early blastocyst, expanded blastocyst. LD were stained with BODIPY 493/503 dye, embryos were visualized under a confocal microscope and images were analyzed with the ImageJ Fiji software. The following parameters were analyzed: lipid content, LD number, LD size and LD area within the total embryo. The most important results show that lipid parameters in the IVF vs. PA bovine embryos differ at the most crucial moments of embryonic development (zygote, 8-16-cell, blastocyst), indicating possible dysregulations of lipid metabolism in PA embryos. When bovine vs. porcine species are compared, we observe higher lipid content around EGA stage and lower lipid content at the blastocyst stage for bovine embryos, which indicates different demand for energy depending on the species. We conclude that lipid droplets parameters significantly differ among developmental stages and between species but also can be affected by the genome origin.

The metabolic profile of bovine blastocysts is affected by in vitro culture system and the pattern of first zygotic cleavage.

Time lapse monitoring (TLM) is a commercial system of individual embryo culture based on the well-of-the-well system. It allows for collecting a panel of intrinsic parameters of special importance to non-invasive quality assessment. Besides, a combined analysis of TLM and metabolome data provides a deeper insight into the embryo quality. Two questions have been addressed by this study: (i) whether embryo culture in the Primo Vision affects embryo development and quality compared to the classical system and (ii) whether the first zygotic cleavage (FZC) dysmorphisms affect metabolomic profile of blastocysts. Presumptive zygotes from IVM/IVF oocytes were cultured either in classical drops (pools of 30 embryos, control group) or on Primo Vision plates (16 embryos in separate wells, sharing culture medium, Primo Vision group). The two categories of blastocysts were analysed for relative transcript abundance of five genes (real time PCR; FASN, Hsp70, PLIN2, Bax, Slc2a1) and for metabolite profile (mass spectrometry). Besides, basing on retrospective analysis of the TLM files, the Primo Vision blastocysts were allocated into one of two groups depending on the FZC pattern (normal NC or direct cleavage DC) and analysed for metabolite profiles. The Primo Vision embryos showed higher cleavage rate (p < 0.01) but reduced blastocyst rate (p < 0.05) when compared to the control group. Although the culture system (Primo Vision vs classical) did not affect the relative transcript abundance, the metabolomic analysis revealed different activity of five pathways. Two of them, related to beta-oxidation of fatty acids were up-regulated, whereas the remaining three corresponding to ubiquinone synthesis, metabolism of phenyloalanine and tyrosine as well as vitamin B6 metabolism were down-regulated. The metabolite profiles were however particularly affected by the FZC pattern. The NC and DC blastocysts differed significantly in the activity of 16 metabolic pathways which mainly involved pyruvic acid. The DC embryos displayed a higher level of pyruvic acid (p < 0.05) which may imply disturbances in the switch from lipid to glucose metabolism. Besides, in our opinion, embryos cultured in separate wells in the Primo Vision may face increased ROS level which reduces their developmental potential. Summarizing we underline application of metabolome analysis to embryo quality assessment due to providing a wider insight into embryo response to environmental factors.

Response of Bovine Cumulus-Oocytes Complexes to Energy Pathway Inhibition during In Vitro Maturation.

Glucose or fatty acids (FAs) metabolisms may alter the ovarian follicle environment and thus determine oocyte and the nascent embryo quality. The aim of the experiment was to investigate the effect of selective inhibition of glucose (iodoacetate + DHEA) or FA (etomoxir) metabolism on in vitro maturation (IVM) of bovine COCs (cumulus-oocyte complexes) to investigate oocyte's development, quality, and energy metabolism. After in vitro fertilization, embryos were cultured to the blastocyst stage. Lipid droplets, metabolome, and lipidome were analyzed in oocytes and cumulus cells. mRNA expression of the selected genes was measured in the cumulus cells. ATP and glutathione relative levels were measured in oocytes. Changes in FA content in the maturation medium were evaluated by mass spectrometry. Our results indicate that only glucose metabolism is substantial to the oocyte during IVM since only glucose inhibition decreased embryo culture efficiency. The most noteworthy differences in the reaction to the applied inhibition systems were observed in cumulus cells. The upregulation of ketone body metabolism in the cumulus cells of the glucose inhibition group suggest possibly failed attempts of cells to switch into lipid consumption. On the contrary, etomoxir treatment of the oocytes did not affect embryo development, probably due to undisturbed metabolism in cumulus cells. Therefore, we suggest that the energy pathways analyzed in this experiment are not interchangeable alternatives in bovine COCs.

Lipid metabolism and developmental potential of mammalian oocytes and embryos / Metabolizm lipidów a potencjal rozwojowy oocytów i zarodków ssaków.

Developmental potential of oocytes and embryos is one of the key factors determining success in reproduction. In vitro produced embryos display reduced quality thus development of non-invasive approaches for quality assessment is a priority. Lipid metabolism belongs to fundamental mechanisms affecting reproductive processes and shaping the quality of gametes and embryos. The cytoplasm of oocytes and embryos contains specialized organelles for lipid storage (lipid droplets) whose number and size is species dependent. The growth and maturation of the oocyte/embryo is accompanied by a great fluctuation in lipid quality and quantity which in turn affects their quality and freezing suitability. There is a possibility to modify lipid parameters both in vivo and in vitro by supplementing fat to diet and culture media. The manuscript presents the current state of knowledge on lipid engagement in the process of quality acquirement by oocytes and embryos of two livestock species ­ cattle and pig.

The effects of dietary medium-chain fatty acids on ruminal methanogenesis and fermentation in vitro and in vivo: A meta-analysis.

The efficacy of methane (CH4 ) suppression using medium-chain fatty acids (MCFA) remains inconclusive, despite a number of studies on this topic are available. We thus carried out a meta-analysis to integrate the published data on different concentrations and types of MCFA such as lauric acid and myristic acid, which investigated ruminal methanogenesis and fermentation in in vitro and in vivo experiments. In vitro MCFA sources were classified either as pure MCFA (lauric acid, myristic acid and their combinations) or as natural MCFA-rich oils (canola oil enriched with lauric acids, coconut oil, krabok oil and palm kernel oil). The MCFA sources used in the in vivo studies were coconut oil, lauric acid, myristic acid and the combination of lauric and myristic acids. A total of 41 studies (20 in vitro and 21 in vivo studies) were compiled in our database, which included the data on CH4 emission, digestibility, ruminal fermentation products and microbial populations. The results showed that the amount of CH4 production per unit of digested organic matter decreased linearly under in vitro conditions (p < .01) and tended to decrease quadratically under in vivo conditions (p < .07) with increasing doses of MCFA. Populations of protozoa (p < .01) in both in vitro and in vivo responded negatively in a linear manner, whereas Archaea population diminished quadratically (p = .04) only in the in vitro conditions with increasing doses of MCFA. Increasing dietary MCFA concentrations also reduced the fibre digestibility linearly (p < .05) in both in vitro and in vivo conditions. CH4 production for different sources of MCFA decreased in following order: coconut oil > lauric acid > myristic acid > mixed lauric and myristic acids > palm kernel oil > canola oil enriched with lauric acids > krabok oil. It can be concluded that the effect of MCFA on ruminal methanogenesis depends on the amount and type of MCFA.

Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq.

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

WNT signalling supported by MEK/ERK inhibition is essential to maintain pluripotency in bovine preimplantation embryo.

Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.

Energy metabolism of follicular environment during oocyte growth and maturation.

Oocyte quality is affected by many factors, among which the environment of growth and maturation seems to be crucial. Studies show that well balanced oocyte energy metabolism has a significant impact on several elements of cytoplasmic and nuclear maturation as well as further embryo developmental competence. Therefore homeostasis between metabolism of glucose and fatty acids in the oocyte is being widely described nowadays. This review aims to discuss the follicular (in vivo) or maturation media (in vitro) environments with regard to glucose and fatty acid metabolism, as the main sources of the energy for the oocyte. A great emphasis is given on the balance between those two metabolic pathways and its further impact on female fertility.

Inhibitor mediated WNT and MEK/ERK signalling affects apoptosis and the expression of quality related genes in bovine in vitro obtained blastocysts.

Culture conditions determine embryo quality, which may be affected on many levels (timing of development, blastomere count, transcripts, metabolite content, apoptosis). Molecular interactions of signalling pathways like MEK/ERK and WNT/ß-catenin are critical for cell-to-cell communication and cellular differentiation. Both pathways are important regulators of apoptosis. We have aimed to verify the prolonged effect of MEK/ERK silencing and WNT activation by chemical inhibitors (2i or 3i systems) on bovine IVP embryos. Apoptotic index, total cell count and transcription of embryo quality markers were evaluated. A higher rate of apoptosis was observed in 2i blastocysts, but was not accompanied by changes in transcript content of genes controlling apoptosis (BAX, BCL2, BAK, BAX/BCL2 ratio). Therefore, alternative pathways of apoptotic activation cannot be ruled out. The expression of genes related to embryo quality (HSPA1A, SLC2A1) was not affected. GJA1 transcripts were significantly higher in 3i blastocysts, what indicates a stimulatory effect of the applied inhibitors on cell-to-cell interactions. The lowest mRNA level of the IFNT2 gene was found in 2i embryos. A variation in the SDHA gene transcript was observed (with the highest content in the 3i blastocysts), what may suggest their reduced quality. It may be concluded that the modifications of culture conditions (activation of the WNT and silencing of the MEK/ERK signalling) might alter pathways crucial for embryo development without causing embryonic death.

The consequences of porcine IVM medium supplementation with follicular fluid become reflected in embryo quality, yield and gene expression patterns.

Oocyte and embryo developmental competence are shaped by multiple extrinsic and intrinsic factors. One of the most extensive research areas in the last decade is the regulation of lipid metabolism in oocytes and embryos of different species. We hypothesized that differences in developmental competence of oocytes and embryos between prepubertal and cyclic gilts may arise due to distinct fatty acid profiles in follicular fluid. We found that supplementation of oocyte maturation media with follicular fluid from prepubertal pigs affected quality and development of embryos from prepubertal pigs while embryos of cyclic pigs were not affected. PLIN2, SCD and ACACA transcripts involved in lipid metabolism were upregulated in embryos originating from oocytes of prepubertal pigs matured with autologous follicular fluid. The surface occupied by lipid droplets tend to increase in oocytes matured with follicular fluid from prepubertal pigs regardless oocyte origin. The change into follicular fluid of cyclic pigs increased the efficiency of embryo culture and improved quality, while gene expression was similar to embryos obtained from cyclic gilts. We assume that the follicular fluids of prepubertal and cyclic pigs influenced the quality of oocytes and embryos obtained from prepubertal pigs which are more susceptible to suboptimal in vitro culture conditions.

Interactions of bovine oocytes with follicular elements with respect to lipid metabolism.

Among many factors, lipid metabolism within the follicular environment emerges as an important indicator of oocyte quality. In the literature a crucial significance is described concerning follicular fluid (FF) composition as well as messenger RNA (mRNA) expression in follicular cells. The aim of this study was to describe the relationship between oocyte, FF and follicular cells with regard to lipid metabolism. The set of data originating from individual follicles comprised: lipid droplets (LD) number in oocytes (BODIPY staining), mRNA expression of seven genes in cumulus and granulosa cells (SCD, FADS2, ELOVL2, ELOVL5, GLUT1, GLUT3, GLUT8; real time polymerase chain reaction) and fatty acid (FA) composition in FF (gas chromatography). Obtained results demonstrate significant correlation between oocyte lipid droplets number and FA composition in FF. However, gene expression studies show significant correlation between LD number and GLUT1 gene only. Moreover, the present experiment revealed correlations between FA content in FF and expression of several genes (SCD, FADS2, ELOVL5, GLUT8) in granulosa cells, whereas only the SCD gene in cumulus cells. We suggest that the results of our experiment indicate the importance of glucose : lipid metabolism balance, which contributes to better understanding of energy metabolism conversion between oocytes and the maternal environment.

Multifactorial analysis of the follicular environment is predictive of oocyte morphology in cattle.

Numerous attempts have been recently made in the search for a reliable, fast and noninvasive assay for selection of oocytes suitable for in vitro embryo production. Potential markers have been described in the follicle such as follicular fluid (FF) or cumulus cells (CCs). However, the reported findings are contradictory, which may reflect the complexity of metabolism of the ovarian follicle. In the present experiment, a data set from individual follicles of known diameter was obtained: cumulus-oocyte complex (COC) morphology, fatty acid composition and glucose concentration in FF as well as apoptotic index in CCs. The obtained data was statistically analyzed either separately (univariate analysis) or simultaneously (multivariate analysis) to examine its predictive value in morphology assessment of bovine COCs. Although the univariate analysis yielded a complex relation system of the selected parameters, no clear outcome could be established. In multivariate analysis, the concentration of the four fatty acids (C16:0, C16:1, C18:1cis9, C22:5n3) and Δ(9)-desaturase (16) as well as elongase activities were selected as covariates. This allowed prediction of the morphology of a COC with an accuracy of 72%, which is the most interesting finding of the experiment. The present study indicates that the multifactorial model comprising of selected parameters related to the follicle appeared more effective in predicting the morphology of a bovine COC, which may improve the effectiveness of in vitro production systems.

Differences in cytoplasmic maturation between the BCB+ and control porcine oocytes do not justify application of the BCB test for a standard IVM protocol.

The Brilliant Cresyl Blue (BCB) test relies on G6PDH activity and a simple protocol for the selection of higher quality oocytes. Although the BCB+ oocytes of all the species that have been investigated are characterized by superior quality when compared to BCB- counterparts, application of the test for embryo production still remains an open issue. The aim of our study was to compare BCB+ and the control oocytes (not subjected to the BCB test) in terms of selected aspects of cytoplasmic maturation (mtDNA copy number, mitochondria distribution, relative transcript abundance of six marker genes). The results of our study revealed more relevant differences within the BCB+ and the control oocytes (before and after IVM) than between the two categories of oocytes. There was no difference in the transcript abundance of the BCB+ and the control oocytes in 5 out of 6 analyzed genes (BMP15, GDF9, ATP5A1, EEF1A, ZAR1) and in mtDNA content (pre-IVM 179609 vs. 176595 and post-IVM 187243 vs. 246984, respectively). With regard to mitochondria distribution in pre- and post-IVM oocytes, there was nonsignificant tendency for a more frequent occurrence of the expected patterns in the BCB+ group. The results of the present study do not support the application of BCB staining in a routine IVM protocol due to relatively high similarity in selected parameters characterizing cytoplasmic maturation of BCB+ and control oocytes. This high similarity may results from the limited amount of less competent BCB- oocytes (10%) still present among nonselected oocytes of proper morphology.

Changes in sub-cellular localisation of trophoblast and inner cell mass specific transcription factors during bovine preimplantation development.

BACKGROUND: Preimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8-16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6. RESULTS: At the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8-16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes. CONCLUSIONS: Cell fate specification in cattle become evident earlier than presently anticipated - around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.

Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro.

Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM.

The quality of porcine oocytes is affected by sexual maturity of the donor gilt.

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 µm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 µm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.

Gilts and sows produce similar rate of diploid oocytes in vitro whereas the incidence of aneuploidy differs significantly.

Oocytes derived from prepubertal gilts show reduced developmental competence when compared to oocytes collected from adult sows. Therefore, the aim of the study was to investigate whether gilts (4-5 months old) and adult sows (average age 3.5 years) of the same breed (Polish Landrace x Polish Large White crossbred) differ with regard to the rate of chromosomally unbalanced oocytes after IVM. COCs derived from individual pairs of slaughterhouse ovaries were matured in vitro and analyzed cytogenetically by conventional staining (Giemsa) and FISH methods (probes corresponding to centromeric regions of pig chromosomes 1 and 10). Altogether, 72 females (31 sows, 41 gilts) and 430 secondary oocytes (194 and 236 oocytes of sows and gilts, respectively) were investigated. Cytogenetic analysis revealed diploid (Giemsa, FISH) and aneuploid (FISH) spreads. The incidence of diploid oocytes was similar for sows (26.0%) and gilts (24.5%) whereas the rate of aneuploid oocytes (nullisomic/disomic) was eight times higher in gilts (10.8%) than in sows (1.3%). Diploid and aneuploid oocytes were observed in 64% of investigated females. Pig chromosome 10 was more frequently disomic/nullisomic compared to chromosome 1 suggesting, that like in human, small porcine chromosomes are often involved in the nondisjunction process. In conclusion, chromosomal imbalance significantly contributes to in vitro embryo production in the pig, since over 60% of females produced diploid or aneuploid gametes. The significantly higher rate of aneuploidy among oocytes derived from gilt ovaries may contribute to the reduced developmental competence of gametes collected from nonmature female pigs.

Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana.

Upon a dark-to-light shift, the conditional fluorescent (flu) mutant of Arabidopsis releases singlet oxygen (1O2) within the plastid compartment. Distinct sets of nuclear genes are activated that are different from those induced by superoxide (O2*-)) and/or hydrogen peroxide (H2O2), suggesting that different types of reactive oxygen species activate distinct signaling pathways. It is not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX). The overexpression of the H2O2-specific scavenger reduced strongly the activation of nuclear genes in plants treated with the herbicide paraquat that in the light leads to the enhanced generation of O2*- and H2O2. In the flu mutant overexpressing tAPX, the intensity of 1O2-mediated cell death and growth inhibition was increased when compared with the flu parental line. Also, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX did not lead to visible stress responses and had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions.