Nobiletin enhances mitochondrial function by regulating SIRT1/PGC-1α signaling in porcine oocytes during in vitro maturation.

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 µM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1α signaling pathway.

Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture.

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.

GRP78 acts as a cAMP/PKA signaling modulator through the MC4R pathway in porcine embryonic development.

Glucose-regulated protein 78 (GRP78) binds to and stabilizes melanocortin 4 receptor (MC4R), which activates protein kinase A (PKA) by regulating G proteins. GRP78 is primarily used as a marker for endoplasmic reticulum stress; however, its other functions have not been well studied. Therefore, in this study, we aimed to investigate the function of GRP78 during porcine embryonic development. The developmental quality of porcine embryos, expression of cell cycle proteins, and function of mitochondria were evaluated by inhibiting the function of GRP78. Porcine oocytes were activated to undergo parthenogenesis, and blastocysts were obtained after 7 days of in vitro culture. GRP78 function was inhibited by adding 20 µM HA15 to the in vitro culture medium. The inhibition in GRP78 function led to a decrease in G proteins release, which subsequently downregulated the cyclic adenosine monophosphate (cAMP)/PKA pathway. Ultimately, inhibition of GRP78 function induced the inhibition of CDK1 and cyclin B expression and disruption of the cell cycle. In addition, inhibition of GRP78 function regulated DRP1 and SIRT1 expression, resulting in mitochondrial dysfunction. This study provides new insights into the role of GRP78 in porcine embryonic development, particularly its involvement in the regulation of the MC4R pathway and downstream cAMP/PKA signaling. The results suggest that the inhibition of GRP78 function in porcine embryos by HA15 treatment may have negative effects on embryo quality and development. This study also demonstrated that GRP78 plays a crucial role in the functioning of MC4R, which releases the G protein during porcine embryonic development.

Knock-down of YME1L1 induces mitochondrial dysfunction during early porcine embryonic development.

YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome c, a pro-apoptotic signal, was released from the mitochondria after the knock-down of YME1L1. Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.

Alpha-lipoic acid attenuates heat stress-induced apoptosis via upregulating the heat shock response in porcine parthenotes.

Heat stress (HS) is a long-standing hurdle that animals face in the living environment. Alpha-lipoic acid (ALA) is a strong antioxidant synthesized by plants and animals. The present study evaluated the mechanism of ALA action in HS-induced early porcine parthenotes development. Parthenogenetically activated porcine oocytes were divided into three groups: control, high temperature (HT) (42 °C for 10 h), and HT + ALA (with 10 µM ALA). The results show that HT treatment significantly reduced the blastocyst formation rate compared to the control. The addition of ALA partially restored the development and improved the quality of blastocysts. Moreover, supplementation with ALA not only induced lower levels of reactive oxygen species and higher glutathione levels but also markedly reduced the expression of glucose regulatory protein 78. The protein levels of heat shock factor 1 and heat shock protein 40 were higher in the HT + ALA group, which suggests activation of the heat shock response. The addition of ALA reduced the expression of caspase 3 and increased the expression of B-cell lymphoma-extra-large protein. Collectively, this study revealed that ALA supplementation ameliorated HS-induced apoptosis by suppressing oxidative and endoplasmic reticulum stresses via activating the heat shock response, which improved the quality of HS-exposed porcine parthenotes.

Y-box binding protein 1 influences zygotic genome activation by regulating N6-methyladenosine in porcine embryos.

Y-box binding protein 1 (YBX1) is a member of the family of DNA- and RNA-binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one-cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four-cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6-methyladenosine (m6A) writer N6-adenosine-methyltransferase 70 kDa subunit (METTL3) and reader insulin-like growth factor 2 mRNA-binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process.

Epigallocatechin-3-gallate protects porcine oocytes against post-ovulatory aging through inhibition of oxidative stress.

Increased levels of oxidative stress are major factors that drive the process of post-ovulatory oocyte aging. Epigallocatechin-3-gallate (EGCG), which accounts for up to 50% of the catechins, possesses versatile biological functions, including preventing or treating diabetes, cancer, and heart diseases. The aim of this study was to explore whether EGCG can delay porcine oocyte aging by preventing oxidative stress. Metaphase II (MII) oocytes were cultured for 48 h with different concentrations of EGCG (0-100 µM) in vitro as a post-ovulatory aging model. An optimal concentration of 5 µM EGCG maintained oocyte morphology and developmental competence during aging. The oocytes were randomly divided into five groups: fresh, 24 h control, 24 h EGCG, 48 h control, and 48 h EGCG. The results suggest that EGCG significantly prevents aging-induced oxidative stress, glutathione (GSH) reduction, apoptosis, and autophagy. Moreover, mitochondria DNA copy number was decreased, and the number of active mitochondria and adenosine triphosphate (ATP) levels significantly increased by supplementation with EGCG. Thus, EGCG has a preventive role against aging in porcine post-ovulatory oocytes due to its ability to inhibit oxidative stress and promote mitochondrial biogenesis.

Lactoferrin: A glycoprotein that plays an active role in human health.

Lactoferrin (Lf), existing widely in human and mammalian milk, is a multifunctional glycoprotein with many functions, such as immune regulation, anti-inflammation, antibacterial, antiviral, and antioxidant. These extensive functions largely attribute to its ability to chelate iron and interfere with the cellular receptors of pathogenic microorganisms and their hosts. Moreover, it is non-toxic and has good compatibility with other supplements. Thus, Lf has been widely used in food nutrition, drug carriers, biotechnology, and feed development. Although Lf has been continuously explored and studied, a more comprehensive and systematic compendium is still required. This review presents the recent advances in the structure and physicochemical properties of Lf as well as clinical studies on human diseases, with the aim of providing a reference for further research of Lf and the development of its related functional products.

Acetyl-CoA synthases are essential for maintaining histone acetylation under metabolic stress during zygotic genome activation in pigs.

ACSS1/2 converts acetate into acetyl-coenzyme A, which contributes to histone acetylation in the mitochondria and cytoplasm. Zygotic genome activation (ZGA) is critical for embryo development involving drastic histone modification. An efficient crRNAs-Cas13a targeting strategy was employed to investigate the ACSS1/2 function during ZGA. The results showed that nuclear accumulation of ACSS1 and ACSS2 occurs during ZGA. Knockdown of ACSS1/2 did not affect blastocyst formation when using a normal medium. On culturing embryos in a medium with acetate and no pyruvate (-P + Ace), knockdown of ACSS1 did not affect histone acetylation levels but significantly reduced ATP levels, whereas knockdown of ACSS2 significantly reduced histone acetylation levels in porcine embryos. Inhibition of fatty acid beta-oxidation by etomoxir significantly reduced ATP levels, which could be restored by acetate. The histone acetylation levels in the ACSS1 and ACSS2 knockdown groups both decreased considerably after etomoxir treatment. Moreover, acetate showed dose-dependent effects on SIRT1 and SIRT3 levels when under metabolic stress. The C-terminus of ACSS1 regulated the nuclear translocation. In conclusion, ACSS1/2 helps to maintain ATP and histone acetylation levels in porcine early embryos under metabolic stress during ZGA.

PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.

Phosphatase and tensin homolog-induced kinase 1 (PINK1) on the outer membranes of impaired mitochondria promotes mitophagy and regulates mitochondrial morphology. Mammalian oocytes and early embryos are mitochondria rich, but mitochondrial dynamics during preimplantation embryo development is not well-studied. To investigate whether PINK1 is required for mitochondrial dynamics in porcine preimplantation embryos, gene knockdown and inhibitors were used, and mitochondrial dynamics were observed by transmission electron microscopy. PINK1 knockdown significantly impaired blastocyst formation and quality, induced mitochondrial elongation and swelling, and reduced mitochondrial DNA copy number. PINK1 knockdown-induced mitochondrial elongation caused mitochondrial dysfunction, oxidative stress, and ATP deficiency, significantly increasing autophagy and apoptosis. Profission dynamin-related protein 1 overexpression prevented PINK1 knockdown-induced impairment of embryo development, mitochondrial elongation, and dysfunction. Thus, PINK1 promotes mitochondrial fission in porcine preimplantation embryos.-Niu, Y.-J., Nie, Z.-W., Shin, K.-T., Zhou, W., Cui, X.-S. PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.

Spindlin1 alters the metaphase to anaphase transition in meiosis I through regulation of BUB3 expression in porcine oocytes.

Spindlin 1 (SPIN1), which contains Tudor-like domains, regulates maternal transcripts via interaction with a messenger RNA (mRNA)-binding protein. SPIN1 is involved in tumorigenesis in somatic cells and is highly expressed in cancer cells. Nevertheless, the role of SPIN1 in porcine oocyte maturation remains totally unknown. To explore the function of SPIN1 in porcine oocyte maturation, knockdown, and overexpression techniques were used. SPIN1 mRNA was identified in maternal stages ranging from GV to MII. SPIN1 was localized in the cytoplasm and to chromosomes during meiosis. SPIN1 knockdown accelerated first polar body extrusion. Oocytes with overexpressed SPIN1 were arrested at the MI stage. SPIN1 depletion caused meiotic spindle defects and chromosome instability. The BUB3 signal was investigated, confirming that SPIN1 affects the stability of Bub3 mRNA as well as BUB3 expression. Further, overexpression of SPIN1 inhibited the degradation and regulation of G2/mitotic-specific cyclin-B1. In summation, SPIN1 regulates the meiotic cell cycle by modulating the activation of the spindle assembly checkpoint.

TIP60 contributes to porcine embryonic development by regulating DNA damage response.

The acetyltransferase TIP60 (also known as Kat5) is a member of the MYST family of histone acetyltransferases and was initially identified as a cellular protein. TIP60 acetylates histone and non-histone proteins and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60 was used to detect the function of TIP60 in porcine parthenogenetic embryos. The results showed that TIP60 inhibition impaired porcine parthenogenetic embryonic development. The mechanism of TIP60 was also determined. We found that the TIP60 inhibition impaired embryonic development by ROS induced DNA damage, as demonstrated by the number of γH2A in the nuclei. TIP60 inhibition triggered DNA damage through the regulation of p53-p21 pathway and TIP60 played a role in DNA repair. TIP60 inhibition decreased the efficiency of DNA repair by regulating 53BP1-dependent repair after DNA damage. Inhibition of TIP60 also increased the adaptive response, autophagy, by modulating LC3. Therefore, TIP60 plays a role in early porcine parthenogenetic embryonic development by regulating DNA damage and repair.

Sodium fluoride exposure exerts toxic effects on porcine oocyte maturation.

Excessive long-term fluoride intake is associated with several health problems, including infertility. However, limited information is available on the toxic effects of fluoride exposure on the female reproductive system, especially oocyte maturation. In this study, we investigated the toxic effect of sodium fluoride (NaF) exposure on porcine oocyte maturation and its possible underlying mechanisms. Our results showed that NaF exposure during porcine oocyte maturation inhibited cumulus cell expansion and impaired polar body extrusion. Cell cycle analysis showed that NaF exposure blocked meiotic resumption, disturbed spindle dynamics, disrupted chromosome separation, and increased aneuploidy in porcine oocytes. Moreover, NaF exposure disturbed mitochondrial function, triggered DNA damage response, and induced early apoptosis in porcine oocytes. NaF exposure also induced oxidative stress, decreased GSH level, and increased cathepsin B activity in and impaired the further development potential of porcine oocytes, as indicated by a decrease in blastocyst formation rate, increase in apoptosis, and inhibition of cell proliferation. Together, these results indicate that NaF exposure impairs the maturation capacity of porcine oocytes by inhibiting cumulus cell expansion, disturbing cytoskeletal dynamics, and blocking nuclear and cytoplasmic maturation, thus decreasing the quality and affecting the subsequent embryonic development potential of porcine oocytes.

TCTP regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality.

If no fertilization occurs for a prolonged time following ovulation, oocytes experience a time-dependent deterioration in quality both in vivo and in vitro due to processes called postovulatory aging. Because the postovulatory aging of oocytes has marked detrimental effects on embryo development and offspring, many efforts have been made to unveil the underlying mechanisms. Here we showed that translationally controlled tumor protein (TCTP) regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality. Spindle dynamics decreased with reduced TCTP level during aging of mouse oocytes. Knockdown of TCTP accelerated the reduction of spindle dynamics, accompanying with aging-related deterioration of oocyte quality. Conversely, overexpression of TCTP prevented aging-associated decline of spindle dynamics. Moreover, the aging-related abnormalities in oocytes were rescued after TCTP overexpression, thereby improving fertilization competency and subsequent embryo development. Therefore, our results demonstrate that TCTP-mediated spindle dynamics play a key role in maintaining oocyte quality during postovulatory aging and overexpression of TCTP is sufficient to prevent aging-associated abnormalities in mouse oocytes.

Spindlin 1 is essential for metaphase II stage maintenance and chromosomal stability in porcine oocytes.

Study question: What is the function of Spindlin 1 (Spin1) in metaphase II stage oocytes in pigs? Summary answer: Depletion of Spin1 induces spontaneous oocyte activation and overexpression of Spin1 causes multinuclear formation through induction of DNA damage in porcine oocytes. What is known already: Little is known about the function of Spin1 in oocytes and embryos. In mouse oocytes, Spin1 is specifically expressed during gametogenesis and is essential for meiotic resumption. In somatic cells, Spin1 promotes cancer cell proliferation and activates WNT/T-cell factor signaling. Study design size, duration: After knockdown (KD) or overexpression of Spin1 in porcine MII-stage oocytes, MII maintenance was checked following additional culture for 24 h. Investigated parthenotes were cultured up to the four cell (72 h) or blastocyst (7 days) stages. Participants/materials, setting, methods: Spin1 was knocked down in porcine oocytes and embryos via microinjection of pig Spin1-targeting siRNA. For Spin1 overexpression, porcine Spin1-eGFP cRNA was generated. Additionally, for rescue experiments, cRNA encoding siRNA-resistant mouse Spin1 was added to the pig Spin1-targeting siRNA. For the overexpression and rescue experiments, microinjection and culture were performed using the same methods as the KD experiments. Main results and the role of chance: KD of Spin1 in MII-stage porcine oocytes reduced metaphase-promoting factor and mitogen-activated protein kinase activities, resulting in spontaneous pronuclear formation without calcium activation. However, the DNA damage response was triggered by Spin1 overexpression, generating the checkpoint protein γH2A.X. Furthermore, Spin1 overexpression blocked metaphase-anaphase transition and led to multinucleation in oocytes and embryos. Large scale data: None. Limitations, reasons for caution: This study is based on in vitro investigations with abnormal expression levels of Spin1. This may or may not accurately reflect the situation in vivo. Wider implications of the findings: Spin1 is essential to maintain MII arrest, but a high level of Spin1 induces DNA damage in oocytes and embryos. Thus, a system to accurately regulate Spin1 expression operates in porcine MII-stage oocytes and embryos. Study funding and competing interest(s): This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2015R1D1A1A01057629). The authors declare no competing financial interests.

Analysis of Cyclin E1 Functions in Porcine Preimplantation Embryonic Development by Fluorescence Microscopy.

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

Inhibition of Fatty Acid Synthase Reduces Blastocyst Hatching through Regulation of the AKT Pathway in Pigs.

Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs.

Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.

Exposure to HT-2 toxin causes oxidative stress induced apoptosis/autophagy in porcine oocytes.

T-2 toxin is a main type A trichothecene mycotoxin which is the most toxic trichothecence. T-2 toxin has posed various toxic effects on human and animals in vigorous cell proliferation tissues like lymphoid, hematopoietic and gastrointestinal tissues, while HT-2 toxin is the major metabolite which is deacetylated by T-2 toxin. In this study, we focused on the toxic effects of HT-2 on porcine oocyte maturation. We treated the porcine oocyte with HT-2 toxin in vitro, and we first found that HT-2 treatment inhibited porcine oocyte polar body extrusion and cumulus cell expansion. We observed the disrupted meiotic spindle morphology after treatment, which might be due to the reduced p-MAPK protein level. Actin distribution was also disturbed, indicating that HT-2 affects cytoskeleton of porcine oocytes. We next explored the causes for the failure of oocyte maturation after HT-2 treatment. We found that HT-2 treated oocytes showed the increased ROS level, which indicated that oxidative stress had occurred. We also detected autophagy as well as early apoptosis in the treatment oocytes. Due to the fact that oxidative stress could induced apoptosis, our results indicated that HT-2 toxin caused oxidative stress induced apoptosis and autophagy, which further affected porcine oocyte maturation.

MLL2 is essential for porcine embryo development in vitro.

Several germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, their function during early embryo development has been poorly explored. In this study, we investigated the role of mixed-lineage leukemia protein 2 (MLL2) in the development of porcine preimplantation embryos. To explore the function of MLL2 in porcine embryo development, expression and localization of MLL2 were assessed by qRT-PCR and immunofluorescence assays. Results showed that expression of MLL2 was significantly reduced after the four-cell stage. However, immunofluorescence results showed that MLL2 only localized in the nucleus of blastocysts, revealing a potential role of MLL2 in regulating the gene expression in the blastocyst stage. Knockdown of Mll2 by double-stranded RNA (dsRNA) caused a reduction in MLL2 signal in porcine blastocyst cells and in blastocyst formation. No significant differences in the cleavage and morula stages were observed. The mechanism of MLL2 regulation in blastocysts was assessed by assaying the trimethylation of histone 3 at lysine 4 (H3K4m3). MLL2 knockdown significantly reduced H3K4m3 in the nucleus and further reduced expression of Sox2 and Magoh genes. In conclusion, MLL2 is essential for porcine embryo development by the regulation of methylation of H3K4 in vitro.