RESUMEN

Calcium ions (Ca2+) regulate cell proliferation and differentiation and participate in various physiological activities of cells. The calcium transfer protein inositol 1,4,5-triphosphate receptor (IP3R), located between the endoplasmic reticulum (ER) and mitochondria, plays an important role in regulating Ca2+ levels. However, the mechanism by which IP3R1 affects porcine meiotic progression and embryonic development remains unclear. We established a model in porcine oocytes using siRNA-mediated knockdown of IP3R1 to investigate the effects of IP3R1 on porcine oocyte meiotic progression and embryonic development. The results indicated that a decrease in IP3R1 expression significantly enhanced the interaction between the ER and mitochondria. Additionally, the interaction between the ER and the mitochondrial Ca2+ ([Ca2+]m) transport network protein IP3R1-GRP75-VDAC1 was disrupted. The results of the Duolink II in situ proximity ligation assay (PLA) revealed a weakened pairwise interaction between IP3R1-GRP75 and VDAC1 and a significantly increased interaction between GRP75 and VDAC1 after IP3R1 interference, resulting in the accumulation of large amounts of [Ca2+]m. These changes led to mitochondrial oxidative stress, increased the levels of reactive oxygen species (ROS) and reduced ATP production, which hindered the maturation and late development of porcine oocytes and induced apoptosis. Nevertheless, after treat with [Ca2+]m chelating agent ruthenium red (RR) or ROS scavenger N-acetylcysteine (NAC), the oocytes developmental abnormalities, oxidative stress and apoptosis caused by Ca2+ overload were improved. In conclusion, our results indicated IP3R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage.

Asunto(s)

RESUMEN

Environmental pollutants, including endocrine disruptors, heavy metals, nanomaterials, and pesticides, have been detected in various ecosystems and are of growing global concern. The potential for toxicity to non-target organisms has consistently been raised and is being studied using various animal models. In this review, we focus on pesticides frequently detected in the environment and investigate their potential exposure to livestock. Owing to the reproductive similarities between humans and pigs, various in vitro porcine models, such as porcine oocytes, trophectoderm cells, and luminal epithelial cells, are used to verify reproductive toxicity. These cell lines are being used to study the toxic mechanisms induced by various environmental toxicants, including organophosphate insecticides, pyrethroid insecticides, dinitroaniline herbicides, and diphenyl ether herbicides, which persist in the environment and threaten livestock health. Collectively, these results indicate that these pesticides can induce female reproductive toxicity in pigs and suggest the possibility of adverse effects on other livestock species. These results also indicate possible reproductive toxicity in humans, which requires further investigation.

Asunto(s)

RESUMEN

P21-activated kinase 1 (PAK1) is a critical downstream target that mediates the effect of small Rho GTPase on the regulation of cytoskeletal kinetics, cell proliferation, and cell migration. PAK1 has been identified as a crucial regulator of spindle assembly during the first meiotic division; however, its roles during the metaphase I (MI) to metaphase II (MII) transition in oocytes remain unclear. In the present study, the potential function of PAK1 in regulating microtubule organization and spindle positioning during the MI-MII transition was addressed in porcine oocytes. The results showed that activated PAK1 was co-localized with α-tubulin, and its expression was increased from the MI to MII stage (p < 0.001). However, inhibiting PAK1 activity with an inhibitor targeting PAK1 activation-3 (IPA-3) at the MI stage decreased the first polar body (PB1) extrusion rate (p < 0.05), with most oocytes arrested at the anaphase-telophase (ATI) stage. IPA-3-treated oocytes displayed a decrease in actin distribution in the plasma membrane (p < 0.001) and an increase in the rate of defects in MII spindle reassembly with abnormal spindle positioning (p < 0.001). Nevertheless, these adverse effects of IPA-3 on oocytes were reversed when the disulfide bond between PAK1 and IPA-3 was reduced by dithiothreitol (DTT). Co-immunoprecipitation revealed that PAK1 could recruit activated Aurora A and transform acidic coiled-coil 3 (TACC3) to regulate spindle assembly and interact with LIM kinase 1 (LIMK1) to facilitate actin filament-mediated spindle migration. Together, PAK1 is essential for microtubule organization and spindle migration during the MI-MII transition in porcine oocytes, which is associated with the activity of p-Aurora A, p-TACC3 and p-LIMK1.

Asunto(s)

RESUMEN

The present study investigated the effects of ellagic acid, a type of polyphenol that does not have a glycan and is composed of four hydroxyl groups and two lactone functional groups, on porcine in vitro fertilization (IVF) by focusing on its anti-hyaluronidase activity. A comparative analysis of ellagic acid and apigenin, which is commonly used as a hyaluronidase inhibitor, was performed. It compared the effects of ellagic acid and apigenin on hyaluronidase activity at different concentrations. The results showed that 10, 20, and 40 µM ellagic acid strongly reduced hyaluronidase activity (P < 0.05). The addition of 20 µM ellagic acid, but not apigenin, to porcine IVF medium effectively reduced polyspermy without decreasing sperm penetration or the formation rates of male pronuclei in cumulus-free oocytes. However, neither ellagic acid nor apigenin affected the number of sperm that bound to zona pellucida (ZP) or the induction of zona hardening and protease resistance. The percentage of acrosome-reacting sperm that bound to the ZP was markedly lower in the presence of 20 µM ellagic acid than in the untreated and apigenin-treated groups, even though the antioxidant capacity of ellagic acid was weaker than that of apigenin. Furthermore, a markedly higher percentage of embryos developed to the blastocyst stage in the ellagic acid-treated group, and the apoptotic indexes of expanded blastocysts produced by the ellagic acid treatment during IVF were significantly low. Therefore, the anti-hyaluronidase effect of ellagic acid markedly suppressed the induction of the acrosome reaction in sperm that bound to the ZP, resulting in a marked decrease in polyspermy under conditions that maintained high sperm penetrability during IVF and sustainment of the developmental potency in porcine oocytes.

Asunto(s)

RESUMEN

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 µM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

Asunto(s)

RESUMEN

Background: As the porcine oocyte is the most sensitive to low-temperature damage, it has been difficult to cryopreserve compared to those from other domestic animals. However, at present, vitrification is used as a method for the cryopreservation of both oocytes and embryos in this species. Aim: Our aim was to analyze alterations in metabolic parameters in vitrified-warmed in vitro matured porcine oocytes at different post-warming recuperation times. In addition, metaphase II plate recovery time analysis, in vitro fertilization, and intracytoplasmic sperm injection were carried out to evaluate oocyte recovery capacity. Methods: Oocytes were vitrified-warmed and then incubated for 0, 3, or 21 hours post-warming to assess biochemical parameters. Results: Oocyte viability and morphology were not affected by vitrification-warming. Cytosolic oxidative status, active mitochondria, and reactive oxygen species levels presented changes at the different time points in control and vitrified-warmed oocytes (p < 0.05) as well as differences between both groups (p < 0.05). Nicotinamide adenine dinucleotide phosphate levels remained constant throughout different recuperation times but were significantly lower in vitrified-warmed oocytes (p < 0.05). Metaphase II plate recovery occurred mostly between 3 and 4 hours post-warming, but the percentage of metaphase II was reduced by vitrification-warming. Sperm head decondensation and pronuclear formation capacities were not modified. Conclusion: In conclusion, vitrification-warming generates biochemical alterations in porcine oocytes that would be, in part, responsible for affecting their performance. Therefore, although the technique is a valid alternative for porcine oocyte cryopreservation, the protocols should be adapted to minimize those alterations.

Asunto(s)

RESUMEN

In vitro embryo production depends on high-quality oocytes. Compared with in vivo matured oocytes, in vitro oocytes undergo precocious meiotic resumption, thus compromising oocyte quality. C-type natriuretic peptide (CNP) is a follicular factor maintaining meiotic arrest. Thus, CNP-pretreatment has been widely used to improve the in vitro maturation (IVM) of oocytes in many species. However, the efficacy of this strategy has remained unsatisfactory in porcine oocytes. Here, by determining the functional concentration and dynamics of CNP in inhibiting spontaneous meiotic resumption, we improved the current IVM system of porcine oocytes. Our results indicate that although the beneficial effect of the CNP pre-IVM strategy is common among species, the detailed method may be largely divergent among them and needs to be redesigned specifically for each one. Focusing on the overlooked role of cumulus cells surrounding the oocytes, we also explore the mechanisms relevant to their beneficial effect. In addition to oocytes per se, the enhanced anti-apoptotic and anti-oxidative gene expression in cumulus cells may contribute considerably to improved oocyte quality. These findings not only emphasize the importance of screening the technical parameters of the CNP pre-IVM strategy for specific species, but also highlight the critical supporting role of cumulus cells in this promising strategy.

Asunto(s)

RESUMEN

In the present study, we attempted to improve the developmental competence of vitrified immature porcine oocytes by the preservation of mitochondrial properties using Cyclosporin A (CsA, inhibitor of mitochondrial membrane permeability transition) and Docetaxel (stabilizer of microtubules, hence mitochondrial distribution). In Experiment 1, Mitotracker red staining revealed reduced mitochondrial activity (MA) in vitrified/warmed oocytes at 0 and 22 h of in vitro maturation (IVM) compared with fresh ones. However, by at 46 h of IVM, MA levels in vitrified oocytes were similar to those in fresh control. Treatment of oocytes with CsA or Docetaxel improved MA at 0 h and 22 h of IVM compared with non-treated vitrified oocytes. However, there were no significant differences among groups in percentages of survival, maturation and embryo development after subsequent IVM and parthenogenetic activation. Nevertheless, a pretreatment with a combination of 10 µg/mL CsA and 0.05 µM Docetaxel improved the blastocyst formation of vitrified oocytes compared with non-treatment counterparts (11.2 ± 1.6% vs 5.9 ± 1.6%, P < 0.05). In conclusion, vitrification reduced mitochondrial activity in GV-stage oocytes during 0-22 h of IVM; however, it was normalized by 46 h IVM. Docetaxel or CsA pretreatment alone did not improve development competence of vitrified oocytes. However, pretreatment with a combination of CsA and Docetaxel could improve blastocyst formation rates.

Asunto(s)

RESUMEN

Imidacloprid (IMI) is an endogenous neonicotinoid insecticide widely used in agriculture and has attracted researchers' attention because of its risks to the environment and human health. Melatonin (MT) is an antioxidant hormone produced by the pineal gland of the brain. Studies have shown that it has a variety of physiological functions and plays a crucial role in the development of animal germ cells and embryos. The potential protective effects of MT against oocyte damage caused by neonicotinoid pesticide toxicity remain unclear. In this study, we report the toxicity of IMI against, and its effects on the quality of, porcine oocytes and the protective effect of MT on IMI-exposed oocytes. The results show that IMI exposure adversely affected oocyte maturation, while MT supplementation ameliorated its toxic effects. Specifically, IMI exposure increased oxidative stress (OS), endoplasmic reticulum stress (ERS), and apoptosis, which may affect polar body expulsion rates and blastocyst formation. Also, IMI exposure reduced oocyte cleavage rates and the number of cells in blastocysts. However, all of these toxic effects can be restored after a melatonin supplementation treatment. In conclusion, these results suggest that melatonin has a protective effect on IMI-induced defects during porcine oocyte maturation.

RESUMEN

Oocyte aging directly affects the subsequent embryonic development. Epifriedelanol is the active ingredient of Aster tataricus L.F. extract, and it possesses potential anti-cancer, anti-inflammatory and antioxidant properties. In addition, epifriedelanol can slow the aging of human skin fibroblasts. To explore the effect of epifriedelanol on the aging of porcine oocytes matured in vitro, the aging model was first established, epifriedelanol was added to in vitro maturation (IVM) medium to investigate its anti-aging effects by observing oocyte maturation and embryonic development potential, and analyzing aging-related gene expression, reactive oxygen species and mitochondrial membrane potential levels. It was found that typical aging of porcine oocytes appeared from 66 h during in vitro maturation. Compared with the 44 h group, a larger perivitelline space, increased abnormality of microtubulin formation, and significantly lower blastocyst rate were observed in the 66 h and 72 h groups. Compared with the 0 µg/mL group, the first polar body extrusion, cleavage and blastocyst rates were significantly improved (P < 0.05) in 10 µg/mL group. The expression of oocyte developmental potential-related, SIRT family-related, antioxidant and anti-apoptotic-related genes was significantly up-regulated (P < 0.05), p53 and pro-apoptotic genes were significantly down-regulated (P < 0.05). In addition, the reactive oxygen species level was significantly decreased (P < 0.01), the mitochondrial membrane potential was significantly elevated (P < 0.01) in 10 µg/mL group. In conclusion, epifriedelanol delays the aging of porcine oocytes cultured in vitro by up-regulating SIRT family gene expression, enhancing the antioxidant and anti-apoptotic capacity of oocytes.

Asunto(s)

RESUMEN

The dynamic balance of Ca2+ in oocytes promotes the recovery of the meiotic arrest phase, consequently promoting oocyte maturation. Hence, the analysis of the maintenance and role of calcium homeostasis in oocytes has important guiding significance for obtaining high-quality eggs and maintaining the development of preimplantation embryos. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are calcium channel proteins that regulate the dynamic balance between the endoplasmic reticulum (ER) and mitochondrial Ca2+. Nevertheless, the expression and role of IP3R in normal pig oocytes have not been reported, and other studies have focused on the role of IP3R in damaged cells. The purpose of this study was to investigate the potential role of IP3R in regulating calcium homeostasis in oocyte maturation and early embryonic development. Our results showed that IP3R1 is stably expressed at different stages of porcine oocyte meiosis, IP3R1 gradually converges to the cortex, and cortical clusters are formed in MII stages. The loss of IP3R1 activity contributeds to the failure of porcine oocyte maturation and cumulus cell expansion, as well as the obstruction of polar body excretion. Further analysis showed that IP3R1 plays an important role in affecting calcium balance by regulating the IP3R1-GRP75-VDAC1 channel between mitochondria and the endoplasmic reticulum (ER) during porcine oocyte maturation. Inhibiting IP3R1 expression-induced ER dysfunction, contributeding to ER calcium concentration ([Ca2+]ER) release outwards into mitochondria and causing mitochondrial free calcium concentration ([Ca2+]m) overload and mitochondrial oxidative stress, which was confirmed by the increase in the level of reactive oxygen species (ROS) and apoptosis. Thereby, IP3R1 plays an important role in affecting calcium balance by regulating the IP3R1-GRP75 -VDAC1 channel between mitochondria and the ER during porcine oocyte maturation, inhibiting IP3R1 expression-induced calcium overload and mitochondrial oxidative stress, and increasing ROS levels and apoptosis.

Asunto(s)

RESUMEN

The generation of genetically modified pigs has an important impact thanks its applications in basic research, biomedicine, and meat production. Cloning was the first technique used for this production, although easier and cheaper methods were developed, such as the microinjection, electroporation, or lipofection of oocytes and zygotes. In this study, we analyzed the production of genetically modified embryos via lipofection of zona-pellucida-intact oocytes using LipofectamineTM CRISPRMAXTM Cas9 in comparison with the electroporation method. Two factors were evaluated: (i) the increment in the concentration of the lipofectamine-ribonucleoprotein complexes (LRNPC) (5% vs. 10%) and (ii) the concentration of ribonucleoprotein within the complexes (1xRNP vs. 2xRNP). We found that the increment in the concentration of the LRNPC had a detrimental effect on embryo development and a subsequent effect on the number of mutant embryos. The 5% group had a similar mutant blastocyst rate to the electroporation method (5.52% and 6.38%, respectively, p > 0.05). The increment in the concentration of the ribonucleoprotein inside the complexes had no effect on the blastocyst rate and mutation rate, with the mutant blastocyst rate being similar in both the 1xRNP and 2xRNP lipofection groups and the electroporation group (1.75%, 3.60%, and 3.57%, respectively, p > 0.05). Here, we showed that it is possible to produce knock-out embryos via lipofection of zona-pellucida-intact porcine oocytes with similar efficiencies as with electroporation, although more optimization is needed, mainly in terms of the use of more efficient vesicles for encapsulation with different compositions.

RESUMEN

In vitro-cultured oocytes are separated from the follicular micro-environment in vivo and are more vulnerable than in vivo oocytes to changes in the external environment. This vulnerability disrupts the homeostasis of the intracellular environment, affecting oocyte meiotic completion, and subsequent embryonic developmental competence in vitro. Glycine, one of the main components of glutathione (GSH), plays an important role in the protection of porcine oocytes in vitro. However, the protective mechanism of glycine needs to be further clarified. Our results showed that glycine supplementation promoted cumulus cell expansion and oocyte maturation. Detection of oocyte development ability showed that glycine significantly increased the cleavage rate and blastocyst rate during in vitro fertilization (IVF). SMART-seq revealed that this effect was related to glycine-mediated regulation of cell membrane structure and function. Exogenous addition of glycine significantly increased the levels of the anti-oxidant GSH and the expression of anti-oxidant-related genes (glutathione peroxidase 4 [GPX4], catalase [CAT], superoxide dismutase 1 [SOD1], superoxide dismutase 2 [SOD2], and mitochondrial solute carrier family 25, member 39 [SLC25A39]), decreased the lipid peroxidation caused by reactive oxygen species (ROS) and reduced the level of malondialdehyde (MDA) by enhancing the functions of mitochondria, peroxisomes and lipid droplets (LDs) and the levels of lipid metabolism-related factors (peroxisome proliferator activated receptor coactivator 1 alpha [PGC-1α], peroxisome proliferator-activated receptor γ [PPARγ], sterol regulatory element binding factor 1 [SREBF1], autocrine motility factor receptor [AMFR], and ATP). These effects further reduced ferroptosis and maintained the normal structure and function of the cell membrane. Our results suggest that glycine plays an important role in oocyte maturation and later development by regulating ROS-induced lipid metabolism, thereby protecting against biomembrane damage.

Production of high-quality gametes is the premise of livestock reproduction and conservation of germplasm resources, especially high-quality oocytes, as oocyte quality determines the quality of offspring. Due to the limitations in approaches and the number of mature oocytes in vivo, in vitro maturation (IVM) culture has become an important way to obtain mature oocytes. However, IVM-cultured oocytes are separated from the follicular microenvironment in vivo and are, thus, more vulnerable than in vivo oocytes to changes in the external environment. Our study was conducted to determine if exogenous supplementation of glycine, the highest content of amino acids in oviduct fluid and follicular fluid, can improve oocyte maturation efficiency in vitro, and analyze the mechanism of glycine. This study demonstrated that glycine can maintain redox balance and block reactive oxygen species-induced lipid peroxidation, thereby protecting against biomembrane damage and reducing the occurrence of ferroptosis to maintain normal oocyte development function. This study will provide a theoretical basis for preventing and improving oxidative damage during oocyte culture in vitro.

Asunto(s)

RESUMEN

Quercetin (QUE) is a component of the flavonoid family that shows various therapeutic properties, such as antioxidant effects. However, whether QUE affects porcine oocyte in vitro aging has not yet been investigated. Therefore, in this study, we applied various doses of QUE to freshly isolated porcine oocytes and found that 10 µM QUE improved the oocyte maturation rate in vitro, as reflected by the increased degree of cumulus cell expansion and first polar body extrusion. More importantly, we found that QUE reduced in vitro aging and improved the maturity level of porcine oocytes after another 24 h of culturing, accompanied by the upregulated expression levels of bone morphogenetic protein 15, growth differentiation factor 9, Moloney sarcoma oncogene, and cyclin-dependent kinase 2. In addition, we found that QUE treatment significantly reduced the intracellular reactive oxygen species levels, apoptosis, and autophagy and upregulated the expression levels of superoxide dismutase 2 and catalase in aged porcine oocytes. In addition, QUE restored impaired mitochondrial membrane potential and spindle assembly in aged porcine oocytes. Our findings demonstrate that QUE can protect porcine oocytes from in vitro aging by reducing oxidative stress and maintaining mitochondrial function.

RESUMEN

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

Asunto(s)

RESUMEN

The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control (p < 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion (p < 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes (p < 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.

RESUMEN

The Ran-GTP/importin ß pathway has been implicated in a diverse array of mitotic functions in somatic mitosis; however, the possible meiotic roles of Ran-GTP/importin ß in mammalian oocyte meiosis are still not fully understood. In the present study, importazole (IPZ), a small molecule inhibitor of the interaction between Ran and importin ß was used to explore the potential meiotic roles of Ran-GTP/importin ß in porcine oocytes undergoing meiosis. After IPZ treatment, the extrusion rate of the first polar body (PB1) was significantly decreased, and a higher proportion of the oocytes were arrested at the germinal vesicle breakdown (GVBD) stage. Moreover, IPZ treatment led to severe defects in metaphase I (MI) spindle assembly and chromosome alignment during the germinal vesicle (GV)-to-MI stage, as well as failure of metaphase II (MII) spindle reassembly and homologous chromosome segregation during the MI-to-MII stage. Notably, IPZ treatment decreased TPX2 expression and abnormal subcellular localization. Furthermore, the expression levels of aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3) were significantly reduced after IPZ treatment. Collectively, these data indicate that the interaction of Ran-GTP and importin ß is essential for proper spindle assembly and successful chromosome segregation during two consecutive meiotic divisions in porcine oocytes, and regulation of this complex might be related to its effect on the TPX2 signaling cascades.

Asunto(s)

RESUMEN

BACKGROUND: Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake, growth, reproduction, and health. Particularly, the germ cells are extremely sensitive to the heat stress. However, the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined. METHODS: Germinal vesicle (GV) porcine oocytes were cultured at 41.5 °C for 24 h to induce heat stress, and then cultured at 38.5 °C to the specific developmental stage for subsequent analysis. Nicotinamide mononucleotide (NMN) was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10 µmol/L, 20 µmol/L, 50 µmol/L or 100 µmol/L, respectively, during heat stress. Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation. RESULTS: Here, we report that NMN supplementation improves the quality of porcine oocytes under heat stress. Specifically, we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles, including the cytoskeleton assembly, cortical granule distribution and mitochondrial function. In addition, heat stress induced the production of excessive reactive oxygen species (ROS) and DNA damage, leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest. More importantly, we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation. CONCLUSIONS: Taken together, our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.

RESUMEN

Reproductive technologies are some of the key directions in the context of the need to preserve and select highly productive farmed animals in terms of economically useful traits. Improvements of the existing models of the in vitro oocyte maturation system help to solve the problem of low yield of porcine embryos at the final stages of preimplantation development. In the present study, a model of culture medium for gametes (NCSU-23 with 10 % homologous follicular fluid, 10 IU hCG and 10 IU eCG) modernized by the addition of 1·106 granulosa cells (GCs) per ml and 0.001 % of highly dispersed silica nanoparticles (HDSn) is proposed for use in the IVM and IVF technology of donor porcine oocytes. Analysis of the oocyte chromatin status by the Tarkowsky method and assessment of the level of destructive changes in chromatin (apoptosis, pyknosis) revealed a significant percentage increase in matured oocytes and a decrease in the proportion of granulosa cells with degenerated chromatin when using the original culture system. The positive effects of a joint addition of GCs and HDSn to the maturation system have made it possible to increase the indicators of the meiotic maturation and fertilization of oocytes. Optimal results of developmental competence of oocytes were achieved with the joint use of GCs and HDSn in the maturation system (the proportion of matured cells reached 89 %, the level of oocytes with chromosome degeneration was 12 %, 39 % of embryos reached the final stage of preimplantation development). The positive effect of HDSn on oocyte fertilization was accompanied by an abrupt decrease in destructive processes in GCs during culture in the presence of HDSn. The level of somatic cells with pyknotic nuclei was 32 % and the level of apoptosis (TUNELtest), 21 %, compared with the control (43 and 31 %, p <0.01, respectively). Thus, a high efficiency of the porcine oocyte maturation system in the joint culture of gametes with GCs and HDSn was revealed. It makes it possible to recommend a model of this culture medium at the IVM of female gametes of Sus scrofa domesticus for improving the quality of donor oocytes used in cell and genetic engineering.

RESUMEN

The targeting protein for Xklp2 (TPX2) is a spindle assembly factor, that can stimulate microtubule formation and promote spindle completion during mitosis. However, the role of TPX2 in mammalian oocyte meiotic maturation is still not fully understood. This study was conducted to address the dynamic distribution and potential roles of TPX2 in microtubule nucleation during meiotic maturation in porcine oocytes by microinjecting specific siRNAs. Western blotting results revealed that the expression of TPX2 displayed a lower level from 0 to 22 h of culture, while its expression exhibited a higher level after 28 h of culture. Immunofluorescence staining demonstrated that TPX2 was distributed along the microtubules and enriched in the poles after meiotic spindle formation at the 28 and 44 h of culture. From immunoprecipitation, TPX2 can interact with the microtubule-associated proteins aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3). Meanwhile, the dynamic changes in the expression and localization of AURKA and TACC3 were highly consistent with TPX2 during meiotic maturation. After knocking down TPX2 by siRNA injection, the proportion of oocytes with aberrant spindles and scattered cytoplasmic actin filaments was significantly increased. In addition, TPX2 depletion markedly downregulated the expression of AURKA and TACC3. Thus, these results suggested that TPX2 is essential for meiotic spindle formation in the porcine oocyte, and that this regulation is related to its effect on AURKA and TACC3 expression.

Asunto(s)